Methods for the identification of agents that modulate the structure and processing of a membrane bound precursor protein

ABSTRACT

The present invention provides methods for the screening and identification of agents from a large library of molecular structures that can alter the cleavage of a membrane protein of interest. Agents identified by the methods of the present invention that modify the cleavage of the membrane protein can be used in the treatment and prevention of diseases such as inflammation, diabetes, cancer, Alzheimer&#39;s disease, Parkinson&#39;s disease, and the like. The methods select for and identify effector agents that bind to the membrane protein of interest causing a structural change in the structure of the membrane protein in such a way that the efficiency of the cleavage of a secretase is modulated. Further, the methods are carried out in an in vivo system that provides for physiological conditions similar or identical to conditions for membrane protein processing. Agents can be selected for their ability to cause a decrease or increase the amount of secretase cleavage of the membrane protein.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/424,030, filed Nov. 4, 2002, incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

Enzymatic conversion, or processing, of cell membrane proteins duringtransit to the membrane or at the cell surface appears to be arelatively common occurrence. For example, a number of membrane proteinsof both Type I and Type II topology also occur as a circulating solubleform. These soluble forms are often derived from a membrane boundprecursor protein by proteolysis. The group of enzymes involved in theseprocesses have become known collectively as “secretases” or “sheddases”.

Typically, cleavage of a membrane bound precursor protein occurs closeto the extracellular membrane surface, releasing the soluble form of theprotein from the cell. This secretion usually involves either a proteaseor a phospolipase depending on the type of membrane anchor on theprotein. Examples of proteins secreted by this mechanism includeangiotensin converting enzyme (ACE; Ramchandran and Sen, Biochemistry34:12645-12652, 1995), β-amyloid precursor protein (APP; Selkoe, TrendsCell Biol. 8:447-453, 1998) and other β-amyloids, transforming growthfactor-α (TGF-α; Massague and Pandiella, Annu. Rev. Biochem. 62:515-541,1993), tumor necrosis factor-α (TNF-α; Black et al., Nature 385:729-733,1997), tumor necrosis factor receptor-I and -II (TNFR-I; Mullberg etal., J. Immunol. 155:5198-5205, 1995, and TNFR-II; Porteu and Nathan, J.Exp. Med. 172:599-607, 1990), Fas ligand (FasL; Tanaka et al., NatureMed. 4:31-36, 1998), Interleukin 6 receptor (IL6R; Mullberg et al., J.Immunol. 152:4958-4968, 1994), and the like. Many of these proteins thatare the product of membrane protein processing are associated withvarious disease states including arthritis, cancer, diabetes,hypertension, Alzheimer's disease, among others.

Modulation of some processing enzymes is currently being examined fortherapeutic agents. In particular, molecules that modulate the activityof a secretase that produces a processing product associated with adisease state are of great interest. For example, tumor necrosisfactor-α converting enzyme (TACE, ADAM17,CD156q) is a member of the “ADisintegrin And Metalloprotease”, or ADAM, family. TACE expression islargely constitutive, but the surface pool of the enzyme appears to bedown regulated following cell activation. Cleavage by TACE generates thesoluble form of tumor necrosis factor-α (TNF-α) that has been associatedwith inflammation. Cell activators increase the rate of cleavage,increasing the amount of TNF-α shedding. Inhibitors of TACE arecurrently being sought to treat inflammatory disease.

In another example, the neuropathology of Alzheimer's disease ischaracterized by the accumulation of extracellular protein deposits inthe brain. These deposits include the amyloid containing plaques andamyloid in vessel walls. The major component of an amyloid plaque is a39-42 amino acid residue self-aggregating peptide called β-amyloid (Aβ).Considerable progress has been made in understanding the mechanisms thatcause the disease, especially following the identification of AmyloidPrecursor Protein (APP) and presenilin (PS) gene mutations in familialforms of Alzheimer's disease. These mutations lead to increased levelsof the Aβ peptide in the brain (Selkoe, Physiological Rev 81:741-766,2001).

The Aβ peptide is a proteolytic fragment of APP, a transmembrane proteinexpressed throughout most tissues in the body. FIG. 2 shows thestructure of APP, the resulting proteolytic fragments, and therespective processing enzymes known to generate these fragments. The APPprotein is processed by at least three different proteases includingα-secretase, β-secretase, and γ-secretase. The β-secretase, responsiblefor the amino-terminal cleavage, was recently identified and cloned(Sinha, et al., Nature 402: 537-540, 1999). This protease preferentiallyreleases Aβ starting at Asp-1 and Glu-11. The γ-secretase cleavage,releasing the carboxyl-termini of APP, apparently occurs in thepredicted transmembrane domain of APP. The γ-secretase consists of acomplex of proteins, the most important being presenilin 1 (Steiner,Rev. Mol. Cell. Biol. 1:217-224, 2000).

A less abundant and more hydrophobic Aβ species, Aβ 1-42, has recentlybeen linked to early pathological changes seen in Alzheimer's disease.Mutations associated with familial forms of Alzheimer's disease havebeen shown to increase the cellular production of Aβ. The Aβ peptide isthus the key molecule in the pathogenic process leading to Alzheimer'sdisease, where Aβ forms protofibrils, fibrils, and subsequently amyloidplaques. Increased production or decreased clearance of Aβ initiates aprocess resulting in amyloid plaque formation and subsequently neuronaldegeneration. Recent studies have suggested the aggregates of Aβ(protofibrils) may be toxic entities contributing to neuronal death(Gotz et al., Science 293:1491-1495, 2001). Hence, any treatment thatlowers Aβ peptide in the brain of Alzheimer patients would be ofsignificant clinical value. FIG. 1 summarizes current knowledge of thedisease in a process called the amyloid cascade.

Numerous research programs are developing drugs that are aimed atreducing the presence of processed products of membrane proteinsassociated with disease, such as Aβ. These programs are primarilyfocused on molecules and agents that function by increasing ordecreasing the activities of the processing enzymes. For example, manyefforts are focused on identifying inhibitors of β-secretase orγ-secretase. However, in neurons, only 5% of APP molecules go throughthe Aβ generating pathway, while 95% of APP molecules are processed byα-secretase, through the non-amyloidogenic pathway, inhibiting Aβformation (Lammish, et al., Proc. Natl. Acad. Sci. USA 96:3922-3927,1999). Another objective is to normalize the production of Aβ byincreasing the level of cleavage at the α-secretase site. Theseapproaches may reduce the formation of the processed product ofinterest, such as Aβ, but may also increase the risk of producing otherhealth related problems due to the involvement of the processing enzymesin the formation of other important biological molecules. For example,increasing the activity of α-secretase reduces the abundance of Aβ.However, α-secretase is also involved in the formation of angiotensinconverting enzyme (ACE), a regulator or blood pressure (Parvathy et al.,Biochemistry 37:1680-1685, 1998).

One approach, with less risk of affecting other important metabolicpathways, is to modulate the abundance of a processing product ofinterest by changing the enzymatic processing of the substrate membraneprotein through a slight alteration in the structure of the substratemembrane protein. For example, studies of APP mutations located close tothe α-secretase cleavage site suggest that local α-helicity contributesto cleavage efficacy presumably by direct interaction of theendoprotease with this structure (Sisodia, Proc. Natl. Acad. Sci. USA89:6075-6079, 1992). Similarly, changes in the amino acid sequence ofAPP identified as the Swedish mutation (Mullan et al., Nature Genetics1:345-347 (1992)), change the structure of APP and increases processingof APP at the β cleavage site.

More specifically, the identification of a molecule (effector) thatmodifies the structure of a protein and consequently the activity of theprotein or its ability to act as a substrate depends on the intrinsicstructural nature of the protein and how it interacts with otherstructures in a biological system. Protein function relies on the aminoacid composition of the protein and the three dimensional structuredictated by the amino acid sequence. A protein is acted upon by otherproteins and structures in a biological system based on its structureand the structure of other interactive molecule(s). The structure of aprotein can be changed by an interaction with another molecule(allosteric interaction), which can increase or decrease the activity ofthe protein or the susceptibility of being acted upon by anotherprotein.

As a particular example, peptides are structures that can interact witha protein to change its structure and consequently the activity of theprotein or its susceptibility to being acted upon by another protein.Peptides as structural effectors are attractive because very largestructural diverse libraries can easily be generated by recombinantmethods and readily screened with procedures that identify preferredphenotypic behavior. These peptide effectors can be used to validate theeffectiveness of a structure to cause a desired structural change. Thepeptide structure can be used as a model compound structure to designand develop, for example, a peptidomimetic structure that would bebiologically stable, readily pass over the blood brain barrier, and besuitable for an oral formulation.

Screening methods have been developed to identify peptides that affectcellular processes through specific binding to proteins (herein alsoreferred to as “peptide effectors”). The utility of random peptidelibraries is demonstrated by the numerous methods that have beendeveloped to generate and screen large libraries of structurally diversepeptides. In addition to chemical strategies, such methods includesystems that rely upon biological generation (see, e.g., Scott andSmith, Science 249:386-390, 1990 (phage display); Kawasaki, U.S. Pat.No. 5,658,754 (in vitro ribosome display); Cull et al., Proc. Natl.Acad. Sci. USA 89:1865-1869, 1992 (random peptide sequences expressed onthe C-terminus of the lac repressor); Murray et al., Biotechnology13:366-372, 1995 (thioredoxin random peptide libraries expressed on theflagellin of E. coli); Brown, Nat. Biotechnol. 15:269-272, 1997(repeating polypeptides expressed on the surface of bacteria); Gilchristand Hamm, Methods Enzymol. 315:388-404, 2000 (peptides-on-plasmids);Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-3755, 2001 (mRNAdisplay); Kjaergaard et al., Appl. Environ. Microbiol. 67:5467-5473,2001 (fimbria-displayed peptide libraries). Most recently, Rigel, Inc.has demonstrated the utility of in vivo introduction of random peptidelibraries to identify peptides that alter phenotypic changes in amammalian cell (See, e.g., Kinsella et al., J. Biol. Chem.277:37512-37518, 2002).

However, current methods do not allow the identification of peptideeffectors that interact with membrane proteins in the cellular secretorypathways and, therefore, are not suited for identification of peptidesthat will affect the processing of the membrane protein duringtransience through the secretory pathways. While some methods have beendeveloped to screen small peptides and polypeptides intracellularly inthe cytoplasm (see, e.g., Fields and Song, Nature 340:245-46, 1989; Cullet al., Proc. Natl. Acad. Sci. USA 89:1865-69, 1992; Lu et al.,Biotechnology 113:366-72, 1995; International Patent Publication WO99/24617; Norman et al., Science 285:591-95, 1999); International PatentPublication WO 98/39483), membrane proteins and their processingenzymes, i.e., APP and APP processing enzymes, can be sequestered incompartments that do not mix with most other cytosolic molecules ororganelles. For this reason, the current methods may not preserve thenative secretory environment of a membrane protein.

In addition, current methods suffer from various disadvantages thatlimit the efficient identification of therapeutically promising peptidesthat act within the extracellular space. Consequently, current methodsare also not suited for efficient identification of peptides that affectthe processing or membrane proteins on the cell surface. Most of thescreening methods that employ conventional peptide libraries (e.g.,phage display libraries, combinatorial libraries, peptide mimeticlibraries, and one-bead-one structure combinatorial libraries)demonstrate only binding to targets in vitro. The normal structure,activity, and any necessary regulatory molecule(s) may be lost whenextracellular proteins are purified or removed from their native,extracellular environment. Thus, these methods often fail to identifypeptides that bind to extracellular targets with correspondingphysiological effects in vivo.

Further, current in vitro screening methods also suffer from thedisadvantage that the normal structure, activity, and any necessaryregulatory molecule(s) may be lost when proteins or other macromoleculesare purified or removed from their native environment. Peptides thatwould normally be effector molecules of native macromolecules may notbind to structurally altered targets. Non-native targets are also morelikely than native targets to non-specifically bind physiologicallyirrelevant peptides. Further, even if purified target molecules retainnative structure and activity, existing screening methods can producepoor or misleading results because the assay conditions are notrepresentative of the local extracellular environment in vivo. Localenvironments can significantly influence the accessibility of targetmolecules to peptides or the specificity or avidity of peptide binding.

Conventional screening methods also typically utilize target moleculesthat are attached to non-physiological surfaces, such as plastic, glass,or polymeric matrices. This association with a non-physiological surfaceintroduces impediments to identifying peptides that interactspecifically with protein or other macromolecular target molecules. Manymacromolecular target molecules that are attached to a non-physiologicalsurface denature onto that surface. Native binding sites on the surfaceof targets can be lost and other sites not normally displayed on thesurface of targets can be unmasked, exposing such physiologicallyirrelevant sites to the peptides. This problem can result in theidentification of peptides that only bind non-specifically to targets.The mode of attachment can also bias how target molecules are exposed onthe surface and can result in a spatial orientation where only one set,or a limited set, of potential binding sites are exposed to thepeptides. When this occurs, functionally important peptide binding siteson targets can be inaccessible during screening. Another impediment isthat the binding kinetics and binding constants of freely soluble,interacting molecules can be altered when one is attached directly to anon-physiological surface. This problem is a well-known phenomenon thatcan either increase or decrease the specificity and avidity of peptidebinding to targets and can lead to the identification of peptides thatare ineffective in subsequent, functional screens (see, e.g.,Vijayendran and Leckband, Anal. Chem. 73:471-480, 2001; Butler, Methods22:4-23, 2000).

Further, chemical-based combinatorial peptide libraries, consisting ofsmall peptides that are not attached to a soluble carrier molecule or ahydrophilic matrix, suffer from the disadvantage that many shortpeptides are not soluble under physiological conditions, such as in thepresence of undiluted blood, plasma, serum, or other complex biologicalfluids. Organic solvents such as methanol, ethanol, or DMSO have beenrequired in prior screens to maintain the solubility of many peptides inthe library. These organic solvents can denature many potential targetor non-target proteins and other macromolecules during screening andresult in the identification of poor-quality peptide candidates.

An additional disadvantage experienced with methods using phage- andbacteria-display peptide libraries is the prevalence of high backgroundsdue to nonspecific binding of phage or bacteria to the targets. Suchbackground can occur when screening is conducted in physiologicenvironments, thereby causing many irrelevant peptide candidates to beselected. Typically, the nonspecific binding of phage and bacteria canbe reduced by screening in the presence of high concentrations of salt,denaturants (e.g., urea or guanidine-HCl), protein, or detergent, orother non-physiological conditions (e.g., elevated temperatures, such asabove 37° C.). In contrast, physiological screening conditions for theidentification of peptides usually replicate the conditions in which thetarget molecules normally express their activities (e.g., human blood at37° C.). However, the complexity of macromolecules (e.g., blood) presentunder physiological conditions can lead to a high level of nonspecificbinding of peptide-displaying phage or bacteria, such that the library'sdiversity can be significantly reduced.

There is a need for screening methods that identify molecular effectorsthat specifically bind to a membrane protein substrate to alter itsprocessing and to modulate the amount of processing product(s), ratherthan identifying agents that generally affect the activities of theprocessing enzymes themselves. The methods should provide for theidentification of molecular effectors that modulate the processing of amembrane protein under conditions that preserve the molecular andcellular constituents of the secretory pathways as well as replicate thecomplex physiological conditions of the extracellular environment.Because maintaining the complex conditions native to those presentduring membrane protein processing will reduce non-specific bindingevents, retain native molecular conformations and kinetics, and maintainthe presence of regulatory molecules, the disclosed physiological andsecretory-based screens are more likely to identify agents that altermembrane protein processing, including agents that alter the productionof a processing product of interest. The present invention provides suchmethods which are further set forth herein.

SUMMARY OF INVENTION

The present invention generally relates to methods for identifying anagent that alters processing of a membrane protein of interest. In oneaspect, the method includes contacting the agent with an animal hostcell that expresses the membrane protein, or a functional fragmentthereof, and at least one processing enzyme of the membrane protein anddetecting an altered processing product on the surface of the host cellto identify the agent that alters the processing of the membraneprotein. Membrane protein processing enzyme(s) expressed by the hostcell can include, for example, a secretase or a sheddase, i.e., aprotease or a phopholipase. The alteration of membrane proteinprocessing detected can be, for example, processing resulting in adecreased production of a soluble form of the membrane protein. Inaddition, the soluble protein demonstrating decreased production as aresult of the altered membrane protein processing can be a solubleprotein associated with an increased risk of disease, such as, forexample, inflammation, diabetes, cancer, Alzheimer's disease,Parkinson's disease, and the like.

The animal host cell expressing the membrane protein and membraneprotein processing enzyme(s) can be, for example, a mammalian host cell.The animal host cell can be a recombinant host cell or, alternatively,an isolated host cell expressing endogenous membrane protein andmembrane protein processing enzyme(s).

In certain embodiments, detecting the altered membrane proteinprocessing includes assessing the relative presence or absence of atleast one species of a processed membrane protein fragment on thesurface of the host cell. As a specific example, species of amyloidprecursor protein (APP) fragments assessed can include, such as, APPs-α,APPs-β, or APPs-γ, can be measured. The assessment of the relativepresence or absence of at least one species of a membrane proteinfragment can include, for example, contacting the host cell with atleast one detectably labeled marker that specifically binds to thespecies of membrane protein fragment and detecting the bound, labeledmarker. Markers for detection of the absence or presence of membraneprotein fragments can be, for example, an antibody that binds to apredetermined epitope of the membrane protein or membrane proteinfragment. In certain embodiments, the assessment of the relativepresence or absence of the membrane protein fragment can includedetermining a ratio of the detection signals of at least two labeledantibodies specific for at least two different epitopes of the membraneprotein or a membrane protein fragment. Detection of altered membraneprotein processing on the surface of the host cell can include, forexample, the use of a flow cytometer or sorter.

The agent contacted with the host cell can be, for example, a smallmolecule or a biomolecule. In certain embodiments, the biomoleculecontacted with the host cell is a peptide. The agent can come from acompound library such as, for example, a combinatorial chemical library,a natural products library, or a peptide library. The agent can be anallosteric effector of the membrane protein.

In certain embodiments, the agent is contacted with the host cell undersubstantially physiological conditions. Substantially physiologicalconditions can include the presence of a complex biological fluid, suchas, for example, blood, serum, plasma, or cerebral spinal fluid (CSF).

In embodiments where the host cell is contacted with a peptide, thepeptide can be produced, for example, by transcription and translationfrom an oligonucleotide encoding the peptide. The length of theoligonucleotides encoding the peptide can be, for example, about 18 toabout 120 nucleotides, about 21 to about 60 nucleotides, or about 36 toabout 60 nucleotides. In one embodiment, the contacting of the peptidewith the host cell includes introducing an expression vector, theexpression vector including the oligonucleotide encoding the peptide,into the host cell. The host cell into which the expression vector isintroduced expresses and displays the peptide within the secretorypathway and on the cell surface.

The oligonucleotides introduced into the host cell can be, for example,from an expression library that includes oligonucleotide inserts, amajority of these oligonucleotides having different sequences encodingdifferent peptides. In certain embodiments, the sequence of theoligonucleotides is randomized. The expression library is introducedinto animal host cells that express the membrane protein and at leastone membrane protein processing enzyme. Host cells into which theexpression library is introduced express and display the differentpeptides within the secretory pathway and on the extracellular cellsurface. In certain embodiments, the different peptides are displayed bythe host cells under substantially physiological conditions.

In other embodiments, a subset of host cells exhibiting altered membraneprotein processing are selected from the host cells. From this subset ofhost cells, a sub-library of the expression library is identified, thesub-library including at least one oligonucleotide that encodes apeptide that alters the processing of the membrane protein.

In certain embodiments, the host cells into which the expression libraryis introduced can be enriched for host cells displaying the differentpeptides. The host cells can be enriched by including a selectablemarker in the expression construct. The expression construct can be, forexample, V5, FLAG, or thioredoxin. Selection for the marker can include,for example, magnetic bead selection fluorescence-activated cellsorting. In certain embodiment, host cells enriched for cell displayingpeptides can express a high copy number of the different peptides.

In one embodiment, the peptide is displayed as a fusion protein with apresentation molecule. The presentation molecule can be, for example,CD24, IL-3 receptor, protein A, or thioredoxin. The fusion protein canfurther include a marker epitope, such as, for example, polyhistidine,V5, FLAG, or myc and the like. The fusion protein can also include asignal for a glycophosphatidylinositol (GPI) anchorage.

In other embodiments, the expression library can be pre-enriched foroligonucleotide(s) encoding peptides that specifically bind to themembrane protein of interest prior to introduction of the library intothe host cells. Pre-enrichment can include introducing the expressionlibrary into a phage display vector which can express the peptidesencoded by the oligonucleotide sequences on the surface of the phage;expressing the different peptides on the surface of the phage; selectinga subset of phage particles that express peptides that specifically bindthe membrane protein; and recovering the oligonucleotide sequences fromthe selected phage particles. The pre-enriched expression library can beintroduced into animal host cells expressing the membrane protein and atleast one membrane protein processing enzyme. Host cells into which apre-enriched library has been introduced express and display themembrane protein-binding peptide(s) within the secretory pathway and onthe extracellular cell surface. A subset of host cells that exhibitaltered membrane protein processing is selected from host cellsexpressing the pre-enriched library. From this subset of host cells, asub-library of the pre-enriched expression library is identified, thesub-library including at least one oligonucleotide that encodes amembrane protein-binding peptide that alters the processing of themembrane protein.

A further understanding of the nature and advantages of the inventionwill become apparent by reference to the remaining portions of thespecification.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the amyloid cascade leading to Alzheimer's disease.

FIG. 2 depicts a schematic diagram of the β-amyloid precursor protein(APP) and its principal metabolic derivatives. The top diagram depictsthe largest of the known APP alternative spliced forms, comprising 770amino acids. A single transmembrane domain (TM) at amino acids 700-723is indicated by vertical dotted lines. The β-amyloid (Aβ) fragmentincludes 28 amino acids outside the membrane plus the first 12-14residues of the TM domain. Arrows indicate sites of the proteolyticcleavage by processing enzymes. The various proteolytic fragments arelabeled. (Selkoe, Physiological Rev. 81:741-767, 2001).

FIG. 3 depicts a representation of the expression cassette for theexpression of random peptide and presentation protein for the retroviralconstruction for the identification of effector peptides of a membraneprotein.

FIG. 4 depicts a representation of the presentation protein beingexpressed on the surface of a cell with random peptide sequence in theconfiguration of a cysteine loop.

FIG. 5 depicts APP on the surface of a cell with labeled fragments,cleavage sites, antibodies recognizing fragments of APP, and strategiesof using ratios of fluorescence to detect altered processing of APP.

FIG. 6 depicts a representation of one example of an expression vector,designated pIcoDual, encoding a CD24 V5 fusion protein and athioredoxin-FLAG fusion protein, which is suitable for use in thepresent invention.

FIG. 7 depicts a representation of an expression vector designatedpIcoFLAGXa, encoding a CD24 V5 fusion protein and a thioredoxin-FLAGfusion protein where the Factor X_(a) restriction cleavage amino acidsequence, Ile-Glu-Gly-Arg-X, SEQ. ID NO:5, has been inserted at therandom peptide site.

FIG. 8 depicts a representation of Thy 1-EMα/EMβ_(s) (S) and Thy1-EMα/EMβ_(M) (M) expression constructs for in vivo expression ofeffector peptides in transgenic mice. “EMα/EMβ sequence” denotes anucleotide coding sequence for either an EMα peptide (i.e., an effectorpeptide that inhibits APP processing by B-secretase).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides screening methods for the identificationof molecules and agents, including peptides and other small molecules,that alter processing of a membrane protein of interest and that therebyalter the production of processed portions of the membrane protein. Themembrane protein of interest is one that is processed either duringtransit to the membrane surface or at the membrane surface. Products ofprocessing of the membrane protein are those that have been associatedwith a disease state, such as inflammation, cancer, diabetes,Alzheimer's disease, Parkison's disease, and the like. The screeningmethods provided allow for the identification of effector agents thatcan increase or decrease cleavage of the membrane protein, or afunctional fragment thereof, at processing enzyme cleavage sites thatcorrelate with the production of a processed product of interest. Thesescreening methods can facilitate the development of therapeuticmolecules for the treatment of inflammatory conditions, cancer,diabetes, Alzheimer's disease (AD), Parkinson's disease, and the like bycorrecting or partially correcting imbalances in the activities ofmembrane protein processing enzymes, such as a secretase, or “sheddase”,that are implicated in the cause of the disease of interest.

Prior to setting forth the invention in more detail, it may be helpfulto a further understanding thereof to set forth definitions of certainterms as used hereinafter.

Definitions

The terms “membrane protein processing enzyme” refers to proteolyticenzymes involved in post-translational modification of a membraneprotein of interest, such as for example, amyloid precursor protein(APP) during transience through secretory pathways in a cell (including,e.g., the trans Golgi network and secretory vesicles) or on the cellsurface. Proteolytic processing of the membrane protein of interestaffects the relative amount of the processed membrane protein producedby the cell. For example, APP processing enzymes include, for example,α-secretase, β-secretase, and γ-secretase.

The term “altered membrane protein processing” refers to a change in therelative amounts of one or more membrane protein fragments produced by acell. APP fragments, for example include, APPs-α, APPs-β, and APPs-γ(see FIG. 2). Because the relative amount of one or more APP fragmentsis correlative of the amount of Aβ produced, “altered APP processing” asused herein also generally refers to changes in APP processing thatresult in a change in the amount of Aβ produced by a cell.

The terms “agent,” “molecule,” and “compound” as used herein aresynonymous and refer generally to molecules potentially capable ofstructural interactions with cellular constituents through non-covalentinteractions, such as, for example, through hydrogen bonds, ionic bonds,van der Waals attractions, or hydrophobic interactions. For example,agents will most typically include molecules with functional groupsnecessary for structural interaction with proteins, glycoproteins,and/or other macromolecules, particularly those groups involved inhydrogen bonding.

Agents can include small organic molecules such as, for example,aliphatic carbon or cyclical carbon (e.g., heterocyclic or carbocyclicstructures and/or aromatic or polyaromatic structures, and the like).These structures can be substituted with one or more functional groupssuch as, for example, an amine, carbonyl, hydroxyl, or carboxyl group.In addition, these structures can include other substituents such as,for example, hydrocarbons (e.g., aliphatic, alicyclic, aromatic, and thelike), nonhydrocarbon radicals (e.g., halo, alkoxy, acetyl, carbonyl,mercapto, sulfoxy, nitro, amide, and the like), or hetero substituents(e.g., those containing non-carbon atoms such as, for example, sulfur,oxygen, or nitrogen).

Agents can also include biomolecules. “Biomolecules” refer to classes ofmolecules that exist in and/or can be produced by living systems as wellas structures derived from such molecules. Biomolecules typicallyinclude, for example, proteins, peptides, saccharides, fatty acids,steroids, purines, pyrimidines, and derivatives, structural analogs, orcombinations thereof. Biomolecules can include one or more functionalgroups such as, for example, an amine, carbonyl, hydroxyl, or carboxylgroup.

Agents include those synthetically or biologically produced and caninclude recombinantly produced structures such as, for example,peptide-presenting fusion proteins. The term “fusion protein” refers toa polymer of amino acids produced by recombinant combination of two ormore sequence motifs and does not refer to a specific length of theproduct; thus, a fusion protein can include a peptide sequence joined toan affinity label such as, for example, 6-histidine.

The terms “effector agent” or “molecular effector” as used herein referto molecules that affect protein interactions with other macromolecules.“Molecular effector of membrane protein processing” thus refers to amolecule that alters the processing of a membrane protein of interestthrough, for example, interactions with the membrane protein orprocessing enzymes or the membrane protein.

The term “specific binding” refers to the direct interaction between anagent and the membrane protein, or a functional fragment thereof. Aninteraction between the agent and the membrane protein, or thefunctional fragment, can be detected either by direct or indirectanalysis.

The term “allosteric effector” refers to an effector agent thatactivates or inhibits a particular protein activity or interaction byspecifically binding to the protein to change its conformation.“Allosteric effector of the membrane protein” thus refers to an agentthat specifically binds to the membrane protein, or a functionalfragment thereof, and changes its conformation such that processing byone or more processing enzyme of the membrane protein is altered. Thespecific binding site of an allosteric effector is referred to herein asan “allosteric site.”

The term “host cell” refers to a cell that can serve as a vehicle totest effector agents that can be introduced by several means. Host cellssuitable for the present invention are those that express the membraneprotein, or a functional fragment thereof, and one or more membraneprotein processing enzymes. In one particular example, the processingenzymes associated with APP include, e.g., α-, β-, and/or γ-secretase.In addition, suitable host cells for use in the present inventiontypically are animal cells, particularly mammalian cells. Host cells canalso be “recombinant host cells.” The term “recombinant host cell” asused herein means a host cells that expresses one or more recombinantproteins, including, for example, recombinant membrane protein, or afunctional fragment thereof, and/or one or more processing enzymes ofthe membrane protein. Examples of suitable host cells include humanembryonic kidney (HEK) cells, human neuroblastoma cell lines, Ba/F3, AC2(see, e.g., Garland and Kinnaird, Lymphokine Res. 5:S145-S150 (1986)),B9, HepG2, MES-SA and MES-SA/Dx5 cells. The host cell can serve as arecipient for a genetic library that is introduced by any one of severalprocedures. The host cell serving as a recipient of a genetic libraryoften allows replication and segregation of a vector containing alibrary insert. In certain embodiments, however, replication andsegregation are irrelevant; expression of a library insert is all thatis required.

The terms “genetic library” refers to a collection of nucleic acidfragments that can individually range in size from about a few basepairs to about a million base pairs. Typically, as used in the contextof the present invention, a genetic library comprises random orsemi-random oligonucleotides that encode peptides or polypeptides. Theoligonucleotides can have an average length of, for example, from about10 bases to about 60 bases. In certain embodiments, a library iscontained as inserts in a vector capable of propagating in certain hostcells, such as bacterial and/or mammalian cells.

The term “compound library” as used herein refers to any collection ofagents that includes a plurality of molecular structures. Compoundlibraries can include, for example, combinatorial chemical libraries,natural products libraries, and peptide libraries, further describedinfra. In certain embodiments, peptide libraries can be generated bytranscription and translation from nucleic acid sequences includedwithin a genetic library.

The term “sub-library” refers to a portion of a compound library orgenetic library that has been isolated by methods according to thepresent invention.

The term “insert” in the context of a genetic library refers to anindividual nucleic acid fragment that is typically inserted into singlevector (e.g. an expression vector) or an expression construct.

The term “coverage” in the context of a genetic library refers to theamount of redundancy of the genetic library. It will be appreciated bythose skilled in the art that the redundancy of a genetic library isgenerally related to the probability that a specific sequence isactually present within the nucleic acid sequences of that library.Coverage is the ratio of the number of library inserts, such aspeptide-encoding oligonucleotides, multiplied by the average insert sizedivided by the total complexity of the nucleic acid sequences that thelibrary represents.

The term “vector” refers to a nucleic acid sequence that is capable ofpropagating in a particular host cell and that can accommodate insertsof heterologous nucleic acid. Typically, vectors are manipulated invitro to insert heterologous nucleic acids into a cloning site. A vectorcan be introduced into a host cell in a stable or transient manner, suchas by transformation, transfection, or infection by a viral vector.

The term “expression vector” refers to a vector designed to express aninserted nucleic acid. Such vectors can contain, for example, one ormore of the following operably associated elements: a promoter locatedupstream of the insertion site (e.g., a cloning site) of the nucleicacid, a transcription termination signal, a translation terminationsignal and/or a polyadenylation signal. An expression vector can alsoinclude a selectable marker, such as a drug resistance gene (e.g.,hygromycin or neomycin resistance). (See, e.g., Santerre et al., Gene30:147-156 (1984)). The expression vector can also include sequences forpackaging into viral particles.

The term “high copy number” refers to expression on an extracellularsurface of a host cell of at least several hundred to several thousandmolecules encoded by a library insert.

The term “expression” in the context of a nucleic acid refers totranscription and/or translation of the nucleic acid into mRNA and/orprotein.

The term “expression library” refers to a plurality of copies of anexpression construct or vector, a majority of the copies of theconstruct or vector containing inserts of nucleic acid fragments fromthe genetic library.

The term “presentation molecule” refers to a polypeptide that can beused to display a peptide or polypeptide as part of a fusion protein.

The term “stable expression” refers to the continued presence andexpression of a nucleic acid sequence in a host cell for a period oftime that is at least as long as that required to carry out the methodsaccording to the present invention. Stable expression can be achieved byintegration of the nucleic acid into a host cell chromosome, orengineering the nucleic acid so that it possesses elements that ensureits continued replication and segregation within the host (e.g., anexpression vector or an artificial chromosome) or alternatively, thenucleic acid can contain a selectable marker (e.g., a drug resistancegene) so that stable expression of the nucleic acid is ensured bygrowing the host cells under selection conditions (e.g., drug-containingmedium) or it can be introduced as a viral genome that becomesintegrated in the host genome.

The term “specific binding” refers to the direct interaction between anagent and the membrane protein. An interaction between the agent and themembrane protein can be detected either by direct or indirect analysis.

The terms “physiological conditions” and “substantially physiologicalconditions” refer to conditions that are normally present, or thatsubstantially approximate those normally present, in an extracellularspace, on an extracellular surface (e.g., on a cell membrane), in aGolgi network, secretory vesicle, and/or in a complex biological fluid.For example, “substantially physiological conditions” can be thosepresent when extracellular targets are active or express theiractivities (e.g., enzymatic activity, binding to a receptor, substrate,scaffolding molecule, or other binding partner, and the like).

The term “complex biological fluid” refers to a biological fluid, suchas, for example, autologous (i.e., from the same animal), homologous(i.e., from an animal of the same species), or heterologous (i.e., froma different species) blood, plasma, serum, cerebral spinal fluid (CSF),and the like. Complex biological fluids can be either undiluted orsubstantially undiluted. The term “substantially undiluted complexbiological fluid” refers to a complex biological fluid that is eitherundiluted or diluted in physiological buffers to typically no less thanabout 50% concentration. Substantially undiluted complex biologicalfluids, i.e., no less than approximately 50% of undiluted fluids, havesubstantially the same ionic composition and strength and substantiallythe same macromolecular structures in solution, in approximately thesame absolute concentrations, as the undiluted fluid.

The terms “transformation” or “transfection” refer to the process ofintroducing nucleic acids into a recipient (e.g., host) cell. This istypically detected by a change in the phenotype of the recipient cell.The term “tansformation” is generally applied to microorganisms, while“tansfection” is used to describe this process in cells derived frommulticellular organisms.

The terms “infect” or “infected” refer to the process of introducingnucleic acids into a recipient (e.g., host) cell by means of a viralvector.

The term “flow sorter” refers to a device that analyzes light emissionintensity from cells or other objects and separates these cells orobjects according to parameters such as light emission intensity.Suitable flow sorters include, for example, a fluorescence-activatedcell sorter (FACS), a spectrophotometer, microtiter plate reader, acharge coupled device camera and reader, a fluorescence microscope, orsimilar device.

The terms “bright” and “dim” in the context of a flow sorter refer tothe intensity levels of fluorescence (or other modes of light emission)exhibited by particular cells: Bright cells have high intensity emissionrelative to the bulk population of cells and, by inference, high levelsof reporter; dim cells have low intensity emission relative to the bulkpopulation and, by inference, low levels of reporter.

The term “bead selection” refers to the use of beads to selectivelyremove cells from a mixture of cells. Beads can include a macromolecule,such as an antibody or other binding partner. In certain embodiments,the bead selection uses derivatized magnetic beads. For example, cellsexpressing a FLAG epitope on the cell surface can be pre-selected onmagnetic beads that are coated with anti-FLAG antibody. The magneticbeads can then be collected using a strong magnetic field.

Selection/Establishment of Host Cell Lines

The host cells used in the screening methods according to the presentinvention express both the membrane protein of interest, for example,TGF-α, TNF-α, APP, TNFR, and the like, and one or more processing enzymeof the membrane protein, such as, in the example of APP, α-secretase,β-secretase, or γ-secretase These host cells can be isolated cells thatendogenously express the membrane protein and/or at least one of theprocessing enzymes of the membrane protein. In addition, the host cellscan be recombinant host cells expressing recombinant forms of themembrane protein, including functional fragments of the membrane proteinthat are properly processed, and/or at least a recombinant form of oneprocessing enzyme. Host cells can, therefore, exhibit endogenousexpression with respect to one membrane protein or enzyme molecule whilebeing a “recombinant host cell” with respect to another such molecule.

In one exemplary embodiment of the invention, the host cells arerecombinant with respect to the membrane protein, such as for example,TGF-α, TNF-α, APP, TNFR, and the appropriate secretase, TGF-α secretase,TNF-α secretase, α-secretase and β-secretase, and TNFR secretase,respectively. DNA encoding the membrane protein of interest can beobtained, for example, from the American Type Culture Collection (ATCC)or the Innovative Molecular Analysis Technologies Program of theNational Cancer Institute, National Institutes of Health (IMAT) or arealternatively obtained by methods known in the art such as, e.g., PCRamplification and DNA sequence analysis verification. The cDNA can beinserted into, e.g., a mammalian expression vector and transfected intoa parental mammalian cell line (e.g., a neuroblastoma cell line or humanembryonic kidney cell line (HEK)) using known methods such as, forexample, electroporation. These cell lines can be assessed forexpression of the membrane protein using methods known in the art suchas, for example, fluorescent microscopy or FACS analysis usingantibodies specific for the membrane protein of interest. For example,antibodies to various APP fragments, including APPs-β (such as forexample, A3 or 1G7 specific to APP midregion, Koo et al., J. Biol. Chem.269:17386-17389, 1994), APPs-α (6E10 specific to the carboxyl terminalend of APPs-α or the region of APP between the β and α cleavage sites,Pirttila et al. Neurol. Sci. 127:90-95, 1994, McLaurin et al., Nat. Med.11:1263-1269, 2002), and p3 (4G8, Pirttila et al. Neurol. Sci.127:90-95, 1994, McLaurin et al., Nat. Med. 11:1263-1269, 2002). Thesecells can then transfected with one or more expression vectors, eachexpression vector encoding one or more APP processing enzymes. Forexample, two separate expression vectors, one encoding α-secretase andthe other encoding either β-secretase or γ-secretase, can be transfectedinto the host cells. DNA encoding the secretases can be obtained, forexample, from the ATCC or IMAT or are alternatively obtained by methodsknown in the art such as, e.g., amplification and DNA sequence analysisverification.

Compound Libraries

In one embodiment of the invention, compound libraries are contactedwith host cells to screen for effector agents that alter processing ofthe membrane protein of interest. Compound libraries can be preparedfrom, for example, a historical collection of compounds synthesized inthe course of pharmaceutical research; libraries of compound derivativesprepared by rational design (see generally, Cho et al., Pac. Symp.Biocompat. 305-316, 1998; Sun et al., J. Comput. Aided Mol. Des.12:597-604, 1998; each incorporated herein by reference in theirentirety), such as, for example, by combinatorial chemistry (seediscussion of combinatorial chemical libraries, infra); natural productslibraries (libraries including, for example, complex extracts derivedfrom microorganisms such as bacteria, algae, fungi, yeasts, molds,various plants or plant parts, animal fluids, secretions and the like,and others, such libraries can, for example, include those formed in thecourse of pharmaceutical research); peptide libraries (see discussion ofpeptide libraries, infra); and the like.

Combinatorial Chemical Libraries: In other embodiments, compoundlibraries can be prepared by syntheses of combinatorial chemicallibraries (see generally DeWitt et al., Proc. Natl. Acad. Sci. USA90:6909-6913, 1993; International Patent Publication WO 94/08051; Baum,Chem. & Eng. News, 72:20-25, 1994; Burbaum et al., Proc. Natl. Acad.Sci. USA 92:6027-6031, 1995; Baldwin et al., J. Am. Chem. Soc.117:5588-5589, 1995; Nestler et al., J. Org. Chem. 59:4723-4724, 1994;Borehardt et al., J. Am. Chem. Soc. 116:373-374, 1994; Ohlmeyer et al.,Proc. Natl. Acad Sci USA 90:10922-10926, 1993; and Longman, Windhover'sIn Vivo The Business & Medicine Report 12:23-31, 1994; all of which areincorporated by reference herein in their entirety.)

The following articles describe methods for selecting starting moleculesand/or criteria used in their selection: Martin et al., J. Med. Chem.38:1431-1436, 1995; Domine et al., J. Med. Chem., 37:973-980, 1994;Abraham et al., J. Pharm. Sci. 83:1085-1100, 1994; each of which ishereby incorporated by reference in its entirety. Methods of makingcombinatorial libraries are known in the art, and include the following:U.S. Pat. Nos. 5,958,792; 5,807,683; 6,004,617; 6,077,954 which areincorporated by reference herein.

A “combinatorial library” is a collection of compounds in which thecompounds of the collection are composed of one or more types ofsubunits. The subunits can be selected from natural or unnaturalmoieties, including dienes, aromatic or polyaromatic compounds, alkanes,cycloalkanes, lactones, dilactones, amino acids, and the like. Thecompounds of the combinatorial library differ in one or more ways withrespect to the number, order, type or types of modifications made to oneor more of the subunits comprising the compounds. Alternatively, acombinatorial library may refer to a collection of “core molecules”which vary as to the number, type or position of R groups they containand/or the identity of molecules composing the core molecule. Thecollection of compounds is typically generated in a systematic way. Anymethod of generating a collection of compounds differing from each otherin one or more of the ways set forth above can be a combinatoriallibrary.

A combinatorial library can be synthesized on a solid support from oneor more solid phase-bound resin starting materials. The library cancontain ten (10) or more, typically fifty (50) or more, organicmolecules which are different from each other (i.e., ten (10) differentmolecules and not ten (10) copies of the same molecule). Each of thedifferent molecules (different basic structure and/or differentsubstituents) will be present in an amount such that its presence can bedetermined by some means (e.g., can be isolated, analyzed, detected witha binding partner or suitable probe). The actual amounts of eachdifferent molecule needed so that its presence can be determined canvary due to the procedures used and can change as the technologies forisolation, detection and analysis advance. When the molecules arepresent in substantially equal molar amounts, an amount, for example, of100 picomoles or more can be detected. Typical libraries includesubstantially equal molar amounts of each desired reaction product andtypically do not include relatively large or small amounts of any givenmolecule(s) so that the presence of such molecules dominates or iscompletely suppressed in any assay.

Combinatorial libraries are generally prepared by derivatizing astarting compound onto a solid-phase support (such as a bead). Ingeneral, the solid support has a commercially available resin attached,such as a Rink or Merrifield Resin. After attachment of the startingcompound, substituents are attached to the starting compound. Forexample, an aromatic (e.g., benzene) compound can be bound to a supportvia a Rink resin. The aromatic ring is reacted simultaneously with asubstituent (e.g., an amide). Substituents are added to the startingcompound, and can be varied by providing a mixture of reactants to addthe substituents. Examples of suitable substituents include, but are notlimited to, the following:

(1) hydrocarbon substituents, that is, aliphatic (e.g., alkyl oralkenyl), alicyclic (e.g., cycloalkyl or cycloalkenyl) substituents,aromatic, aliphatic and alicyclic-substituted aromatic nuclei, and thelike, as well as cyclic substituents;

(2) substituted hydrocarbon substituents, that is, those substituentscontaining non-hydrocarbon radicals which do not alter the predominantlyhydrocarbon substituent; those skilled in the art will be aware of suchradicals (e.g., halo (especially chloro and fluoro), alkoxy, mercapto,alkylmercapto, nitro, nitroso, sulfoxy, and the like);

(3) hetero substituents, that is, substituents that will, while havingpredominantly hydrocarbyl character, contain other than carbon atoms.Suitable heteroatoms will be apparent to those of ordinary skill in theart and include, for example, sulfur, oxygen, nitrogen, and suchsubstituents as pyridyl, furanyl, thiophenyl, imidazolyl, and the like.

Natural Products Library: In another embodiment of the invention, thecompound library is a natural products library. The natural productslibrary can be, for example, a library of natural products from diversenatural products sources (e.g. such as those natural productsaccumulated during the course of pharmaceutical research) or,alternatively, a collection of compounds derived from a single naturalproducts source (for example, one or more lysates, homogenates, orchemical extracts from, e.g., microorganisms, plants, animal fluids, orother biological material such as that found in, e.g., soil or peat).

In one embodiment, the source for generating the natural productslibrary is a peat material. These materials commonly contain very largenumbers of diverse compounds. In one specific embodiment, the naturalproducts library is derived from a peat material obtained from BonaparteMeadows, a peat bog near Bonaparte Lake, Wash., U.S.A. (See U.S. Pat.No. 6,267,962.) Procedures related to the use and screening of peatmaterial for certain uses are generally known in the art. (See, e.g.,U.S. Pat. Nos. 6,267,962 and 6,365,634, incorporated herein by referencein their entirety.) For example, one general scheme of peat materialextraction and fractionation involves an initial exposure to ethanol toextract molecules with a broad range of characteristics from non-polarto polar properties. Subsequent fractions can include, for example,those that are acidified or alkalinized and subjected to phaseseparations with, e.g., chloroform. Resulting fractions can be furtherfractionated by, e.g., silica gel chromatography and/or reverse phaseHPLC. Once a desired fraction is obtained, it can be buffer exchangedusing, e.g., standard procedures to facilitate its use according theparticular screening method used.

Peptide Libraries: In one embodiment, compound pools can be preparedfrom peptide libraries. Generally, peptides ranging in size from about 4amino acids to about 100 amino acids can be used, with peptides rangingfrom about 6 to about 40 being typical and with from between about 7 and12 amino acids to about 20 being more typical.

In some embodiments, the library can comprise synthetic peptides. Forexample, a population of synthetic peptides representing all possibleamino acid sequences of length N (where N is a positive integer), or asubset of all possible sequences, can comprise the peptide library. Suchpeptides can be synthesized by standard chemical methods known in theart (see, e.g., Hunkapiller et al., Nature 310:105-111, 1984; Stewartand Young, Solid Phase Peptide Synthesis, 2^(nd) Ed., Pierce ChemicalCo., Rockford, Ill., (1984)), such as, for example, an automated peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be used in substitution of or inaddition into the classical amino acids. Non-classical amino acidsinclude but are not limited to the D-isomers of the common amino acids,α-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid,γ-amino butyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,selenocysteine, fluoro-amino acids, designer amino acids such asβ-methyl amino acids, C α-methyl amino acids, N α-methyl amino acids,and amino acid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

In other embodiments, the peptide libraries can be produced bytranscription and translation from a library of nucleic acid sequences.In one exemplary embodiment, an expression library comprisingoligonucleotides encoding the library peptides is introduced into a hostcell (see Genetic Libraries, Expression Cassettes and Vectors, andNucleic Acid Transfer, infra).

Genetic Libraries

In one aspect of the invention, the screening methods includeintroducing an expression library into a host cell that expresses themembrane protein of interest, or a function fragment thereof, such asTGF-α, TNF-α, APP, TNFR, and the like, and one or more processingenzyme(s) of the membrane protein.

The genetic libraries according to the present invention include acollection of at least partially heterogeneous nucleic acid fragments.Such nucleic acid fragments can include, for example, synthetic DNA orRNA, genomic DNA, cDNA, mRNA, cRNA, heterogeneous RNA, and the like. Thenucleic acid fragments can represent, for example, all or some portionof a population of nucleic acids, such as a genome, of a population ofmRNAs, or some other set of nucleic acids that contain nucleic acidsequences of interest. The genetic libraries contain sequences in a formthat can be manipulated.

The present invention typically uses genetic libraries that are derivedfrom synthetic DNA or from fragments of genomic DNA and/or cDNA from aparticular organism. Such library sequences will typically range fromabout 10 bases to about 10 kilobases. The library sequences canoptionally be oligonucleotides having, for example, an average length offrom about 10 bases to about 60 bases.

Methods of making synthetic DNA are known to those of skill in the art.(See, e.g., Glick and Pasternak, Molecular Biotechnology: Principals andApplications of Recombinant DNA, ASM Press, Washington, D.C. (1998).)Methods of making randomly sheared genomic DNA and/or cDNA, and ofmanipulating such DNA's, are also known in the art. (See, e.g., Sambrooket al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold SpringHarbor Publish., Cold Spring Harbor, N.Y. (2001); Ausubel et al.,Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons,New York (1999); which are incorporated by reference herein.) Thedetails of library construction, manipulation and maintenance are alsoknown in the art. (See, e.g., Ausubel et al., supra; Sambrook et al.,supra.)

In some aspects, the library is made of synthetic nucleic acidfragments. For example, a population of synthetic oligonucleotidesrepresenting all possible sequences of length N (where N is a positiveinteger), or a subset of all possible sequences, can be the nucleicacids for the library. A population of synthetic oligonucleotidesencoding all possible amino acid sequences of length N, or a subset ofall possible sequences, can also be the nucleic acids for the library.Alternatively, a semi-random library can be used. For example, asemi-random library can be designed according to the codon usagepreference of the host cell or to minimize the inclusion oftranslational stop codons in the encoded amino acid sequence. As anexample of the latter, in the first position of each codon, equimolaramounts of C, A, and G and a one half-molar amount of T would be used.In the second position, A is used at a one half-molar amount while C, T,and G would be used in equimolar amounts. In the third position, onlyequimolar amounts of G and C would be used.

Synthetic oligonucleotides can optionally include any suitable cisregulatory sequence, such as, for example, a promoter, a translationalstart codon, a translational termination signal, a transcriptionaltermination signal, a polyadenylation signal, a cloning site (e.g., arestriction enzyme sites or cohesive end(s)), a sequence encoding anepitope, and/or a priming segment. For example, a library can includeDNA fragments having a restriction enzyme site near one end, operablyassociated with an ATG start codon, a random or semi-random sequence ofN nucleotides, a translational stop codon, a primer binding site and arestriction enzyme site at the other end. Such a collection of fragmentscan be directly ligated into an expression construct, into a vector,into an expression vector, and the like. The fragments can be introducedas single stranded or double stranded DNA, and as either sense orantisense strands. As will be appreciated by the skilled artisan, doublestranded nucleic acids can be formed, for example, by annealingcomplementary single stranded nucleic acids together or by annealing acomplementary primer to the nucleic acid and then adding polymerase andnucleotides (e.g., deoxyribonucleotide or ribonucleotide triphosphates)to form double stranded nucleic acids. Double stranded nucleic acids canalso be formed by ligating single stranded nucleic acids (e.g., DNA)into a site with 5′ and 3′ overhanging ends and then filling in thepartially single stranded nucleic acids with a polymerase and nucleotidetriphosphates. The details of manipulating and cloning oligonucleotidesare known in the art. (See, e.g., Ausubel et al., supra; Sambrook etal., supra.)

The libraries most typically comprise nucleic acids that have coveragethat exceeds the possible permutations of the nucleic acid of thelibrary sequences. For example, a library can comprise a number ofnucleic acids that exceeds the possible permutations of nucleic acidsequences by about 5 times, although greater and lesser amounts ofredundancy are within the scope of the invention. The details of libraryconstruction, manipulation and maintenance are known in the art. (See,e.g., Ausubel et al., supra; Sambrook et al., supra.)

In an exemplary embodiment, a library is created according to thefollowing procedure using methods that are well known in the art. Doublestranded DNA fragments are prepared from random or semi-random syntheticoligonucleotides, randomly cleaved genomic DNA and/or randomly cleavedcDNA. These fragments are treated with enzymes, as necessary, to repairtheir ends and/or to form ends that are compatible with a cloning sitein an expression vector. The DNA fragments are then ligated into thecloning site of copies of the expression vector to form an expressionlibrary. The expression library is introduced into a suitable hoststrain, such as an E. coli strain, and clones are selected. The numberof individual clones is typically sufficient to achieve reasonablecoverage of the possible permutations of the starting material. Theclones are combined and grown in mass culture, or in pools, forisolation of the resident vectors and their inserts. This process allowslarge quantities of the expression library to be obtained in preparationfor subsequent procedures described herein.

Expression Cassettes and Vectors

In another aspect, expression cassettes and/or vectors are used toexpress peptides and/or fusion proteins encoded by sequences of anexpression library. There are numerous expression cassettes and vectorsknown in the art which are readily available for use. (See, e.g.,Ausubel et al., supra; Sambrook et al., supra.) Some of these cassettesand vectors are tailored for use in specific cell types, while otherscan be used in a wide variety of cell types. In mammalian cells, viraltranscriptional regulatory elements are a typical choice for drivingexpression of exogenous coding sequences, such as library sequences. Anexpression cassette or vector can also include one or more selectablemarkers to identify host cells that contain the expression vector and/orthe expression library.

To effect expression of peptides, an expression cassette can include,for example, in a 5′ to 3′ direction relative to the direction oftranscription, a promoter region operably associated with a cloning sitefor insertion of library sequence and a transcriptional terminationregion, optionally having a polyadenylation (poly A) sequence. Theexpression cassette can optionally include a ribosome binding sequence,a translation initiation codon, and/or a translational terminationcodon. A secretion signal and/or a domain for anchoring the expressedpeptide to the cell surface are typically included adjacent the cloningsite.

Suitable secretion signals include, for example, those from CD24.Suitable cell surface-anchoring domains include, for example, a signalfor glycophosphatidylinositol (GPI) anchorage or a transmembrane domain(e.g., the transmembrane domain of CD24, IL-3 receptor, and the like).

To effect expression of the library sequences in host cells of aparticular type, a promoter capable of conferring robust, high ormoderately high expression of the library insert is preferred. Suitablepromoter sequences can include, for example, enhancer and/or a TATA boxsequences capable of binding an RNA polymerase (such as RNA polymeraseII). The promoter can be constitutively active (such as a viralpromoters), or it can be inducible. An inducible promoter can be usedwhen controlled expression of library sequences is desired and/or toavoid toxic side affects associated with expression or over-expressionof peptide sequences and/or fusion proteins. Suitable induciblepromoters include, but are not limited to, interferon inducible promotersystems, the promoters for 3′-5′ poly (A) synthetase or Mx protein (see,e.g., Schumacher et al., Virology 203:144-148, 1994), the HLV-LTR, themetallothionen promoter (see, e.g., Haslinger et al., Proc. Natl. Acad.Sci. USA 82:8572-8576, 1985), the SV40 early promoter region (Bernoistand Chambon, Nature 290:304-310, 1981), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell22:787-797, 1980), the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. USA 78:1441-1445, 1981), and the like.

Other suitable promoters can be derived from housekeeping genes that areexpressed at high or reasonably high levels. For example, the promoterfor β-actin is useful for high expression. (See, e.g., Qin et al., J.Exp. Med 178:355-360, 1993.) Similarly, the cytomegalovirus promoter andthe translational elongation factor EF-1α promoter are other strongpromoters useful for expression. In general, suitable promoters, such ashousekeeping or viral gene promoters, can be identified using well knownmolecular genetic methods.

In certain embodiments, the cloning site is adjacent to one or moretranslational termination sequences, such that the length of anyresulting expressed peptide is substantially the same as the codingregion of the library sequence. As used herein, the phrase“substantially the same length” means that the length of the expressedpeptide corresponds to the length of the coding region in the librarysequence and can further encode, for example, a methionine residuecorresponding to the start codon, any additional amino acids resultingfrom linker nucleic acids within the coding region, translational orpost-translational modifications, one or more epitopes, and the like.

In some embodiments, the cloning site is flanked by epitopes. Suitableepitopes can include, for example, Xpress™ leader peptide(Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys, SEQ ID NO: 1; InVitrogen), a mycepitope (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-Asn, SEQ ID NO:2;InVitrogen), the V5 epitope(Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr; SEQ ID NO:3),the FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, SEQ ID NO:4, (see, e.g.,Hopp et al., Biotechnology 6:1205-1210, 1988), the lexA protein,thioredoxin, FLAG, polyhistidine, and the like.

In an exemplary embodiment, to lend additional structure to theexpressed peptide sequences, the cloning site can be flanked bysequences encoding cysteines such that the expressed peptide willinclude cysteine residues at the termini.

In certain embodiments, a cloning site is associated with the codingregion of a fusion protein for extracellular display of the peptides(also referred to as a “presentation molecule”). Such a fusion proteincan include, for example, (1) homologous protein domains, proteinfragments, or proteins as found in the host cell or on the host cellsurface, and/or (2) heterologous protein domains, protein fragments, orproteins from another type of cell. The choice of fusion protein dependson the type of host cell(s), the stability of the fusion protein, thedesired conformation of the expressed peptide (e.g., constrained orunconstrained). Such a presentation molecule typically includes a signalsequence and a transmembrane domain.

The presentation molecule can display the peptide at or near theN-terminus, at or near the C-terminus, or internally to the presentationmolecule. In an exemplary embodiment, the presentation molecule displaysthe peptide at the N-terminus and the C-terminal portion of thepresentation molecule is anchored to the cell membrane by atransmembrane domain or GPI anchor. The presentation molecule can bemodified to position the peptides at varying distances from the hostcell surface to increase the probability of achieving the appropriatesteric orientation for specific binding between peptides and themembrane protein of interest. In addition, spacers (e.g., glycinespacers) can be included between the presentation molecule and thepeptide to impart flexibility and minimize steric hindrance from thepresentation molecule with peptide interactions in the extracellularspace or in the secretory pathway, including interactions of the peptidewith the membrane protein or the processing enzymes of the membraneprotein. Such spacers can also be included between the presentationmolecule and the cell surface-anchoring domain to impart flexibility atthe cell surface.

Suitable presentation molecules can include, for example, lymphocyteantigen CD20, modified IL-3 receptor, CD24 (see, e.g., Poncet et al.,Acta Neuropathol. (Berl) 91:400-408, 1996), protein A, and the like.Referring to FIG. 6, the pIcoDual vector includes exemplary expressioncassettes. For example, one expression cassette encodes a CD24-V5 fusionprotein and includes one or more unique restriction sites for insertionof library sequences. Another suitable fusion protein includes E. colithioredoxin and the FLAG epitope. At the junction between thethioredoxin and FLAG coding sequences, a unique XbaI restriction sitepermits insertion of library sequences into the fusion protein codingregion.

An expression cassette can optionally be part of an expression vector.Suitable expression vectors are known in the art. (See, e.g., Ausubel etal., supra; Sambrook et al., supra.) In certain embodiments, acontrolled plasmid amplification system is used for expression inmammalian cells. Such a system allows controlled plasmid amplificationin a variety of cells. Increased plasmid copy number can also lead toincreased expression of the encoded peptides. High level expression ofthe peptides can increase the numbers of peptides displayed on anextracellular surface. Such a controlled amplification system alsoallows for sustained transient expression in mammalian cells. Sustainedtransient expression can be advantageous because typically 10 times asmany cells exhibit transient expression as compared to stabletransfection which can allow larger numbers of peptides to beeffectively screened. Plasmid amplification also facilitates recovery ofplasmids or sequences encoding peptides of interest.

In an exemplary embodiment, the controlled plasmid amplification systemutilizes the SV40 replication system. The expression vector contains afusion of the early promoter of SV40 and the coding region for Large Tantigen, so that transcription of Large T antigen is under the controlof the early promoter of SV40. The vector also contains the SV40 originof replication. When this vector enters a cell, the SV40 early promoterpromotes transcription of Large T antigen RNA. The RNA is translatedinto Large T antigen. Large T antigen binds to the SV40 origin and causeamplification of the plasmid. As Large T antigen concentrations rise inthe cell, the binding of Large T antigen to the SV40 early promotershuts down the SV40 early promoter and, consequently, Large T antigenRNA synthesis. The system, therefore, is self-regulating. As the plasmidcopy number rises, there will not be an increase in production of LargeT antigen that would continue to escalate plasmid amplification to thepoint of cell death. The amount of Large T antigen in a cell will be afunction of the amount of Large T antigen RNA, the stability of theLarge T antigen RNA, the stability of Large T antigen protein, therelative affinity for the origin of replication and the SV40 earlypromoter, and the reduction in the amounts of vector, Large T antigenRNA, and Large T antigen due to cell division. Because the amplificationsystem is contained on a vector, plasmid amplification is typically notlimited to the use of COS7 host cells, but rather plasmid amplificationcan be used for most mammalian cell types.

For other replication systems, the expression vector, if it is of viralorigin, may not require propagation in a bacterial host. More typically,however, the vector is propagated in a bacterial host and containssequences necessary for replication and selection in E. coli, such as,for example, a colE1 replicon and an antibiotic resistance gene.

An expression vector can optionally contain one or more selectablemarkers. For example, suitable selectable markers for transfection ofeukaryotic cells include the genes for hygromycin resistance, neomycinresistance, blasticidin resistance, zeocin resistance, doxorubicinresistance, and the like. Suitable selectable markers for other cellsinclude other antibiotic resistance genes and those complementingauxotrophies (e.g., amino acid auxotrophies). The expression vector canalso optionally include a selectable marker to signal that the host cellcontains the expression vector. Suitable selectable markers will includegreen fluorescent protein, or epitopes such as, for example,polyhistidine, the Xpress™ leader peptide(Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys, SEQ ID NO: 1; InVitrogen), a mycepitope (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-Asn, SEQ ID NO:2;InVitrogen), the V5 epitope(Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr; SEQ ID NO:3),the FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, SEQ ID NO:4, see, e.g.,Hopp et al, Biotechnology 6:1205-1210, 1988), the lexA protein, orbacterial thioredoxin. Such markers can be detected, for example, byenzyme assay, by fluorescence using a flow sorter or similar device,using antibodies (e.g., a monoclonal or polyclonal antibody), using beadselection, and the like. When such markers are present on the cellsurface, they can be used to isolate or to enrich for cells expressingthe marker.

Nucleic Acid Transfer

A variety of methods can be used to transfer library sequences into hostcells. (See generally Ausubel et al., supra; Sambrook et al., supra.)Some methods give rise primarily to transient expression in host cells(i.e., the expression is gradually lost from the cell population). Othermethods can generate cells that stably express the library sequences,though the percentage of stable expressers is typically lower thantransient expressers. Such methods include viral and non-viralmechanisms for nucleic acid transfer.

Suitable mammalian cells include, for example, human embryonic kidney(HEK) cells, human neuroblastoma cell lines, K562, COS7, Ba/F3, AC2(see, e.g., Garland and Kinnaird, Lymphokine Res. 5:S145-S150, 1986),B9, HepG2, MES-SA, MES-SA/Dx5 cells, and the like. Animal host cells caninclude, but are not limited to, cells isolated from oncogenic tissuesand tumors, including melanocyte, colon, prostate, leukocytes, liver,kidney, uterus, and the like. Certain cells and cell lines are availablecommercially from, for example, the American Type Culture Collection.

For viral vectors, the library sequences are typically carried into thehost cell as part of the viral package. Depending on the type of virus,the nucleic acid can remain as an extrachromosomal element (e.g.,adenoviruses (see, e.g., Amalfitano et al., Proc. Natl. Acad. Sci. USA93:3352-3356, 1996) or adeno-associated virus) or it can be incorporatedinto a host chromosome (e.g., retroviruses (Iida et al., J. Virol.70:6054-6059, 1996)).

For the transfer of non-viral expression vectors, many methods can beused. (See, e.g., Ausubel et al., supra; Sambrook et al., supra.) Onemethod for nucleic acid transfer is calcium phosphate coprecipitation ofnucleic acid. This method relies on the ability of nucleic acid tocoprecipitate with calcium and phosphate ions into a relativelyinsoluble calcium phosphate complex, which settles onto the surface ofadherent cells on the culture dish bottom. Other methods employlipophilic cations that bind nucleic acid by charge interactions whileforming lipid micelles. These micelles fuse with cell membranes,introducing the nucleic acid into the host cell where it is expressed.Another method of nucleic acid transfer is electroporation, whichinvolves the discharge of voltage from the plates of a capacitor througha buffer containing nucleic and host cells. This process disturbs thecell membrane sufficiently that nucleic acid contained in the buffer isable to penetrate those membranes. Another method involves usingcationic polymers, such as DEAE dextran, to mediate nucleic acid entryand expression in cultured cells. Another method employs ballisticdelivery of nucleic acid into cells. Finally, microinjection of nucleicacid can be used.

Large numbers of identical vectors (e.g., expression vectors containinglibrary sequences) can be introduced into each animal cell by fusingsuch cells with spheroplasts of bacteria harboring a multi-copy vector.The fusion is performed in a manner that on the average allows for thefusion of one spheroplast with one animal cell. For example, when a highcopy number plasmid, such as a derivative of a pUC plasmid, is used,many identical plasmids are typically introduced into each animal cell.This method circumvents the need for amplification of the vector inanimal host cells, and allows for high copy number in the host cells andthe resulting high levels of expression of library sequences. Thisprocedure can also provide for longer periods of transient expressionwithout a need to amplify the vectors in animal host cells. High copynumbers of vector also increase the ease with which library sequencescan be recovered from animal host cells which exhibit a change inreporter expression.

In some of these methods, multiple nucleic acids which can encodepolypeptides which might interact with a target molecule are introducedinto individual cells. Methods are known in the art to minimize transferof multiple fragments. For example, by using “carrier” nucleic acid(e.g., DNA such as salmon or herring sperm DNA, tRNA, and the like), orby reducing the total amount of nucleic acid applied to the host cells,the problem of multiple fragment entry can be reduced. In addition, eachrecipient cell can receive multiple nucleic acid fragments. Multiplepassages of the library through the host cells permit sequences ofinterest to be separated ultimately from other sequences that can bepresent initially as false positives.

In a preferred embodiment, retroviral vectors introduce one peptidesequence into each cell. This supplies a robust signal and reduces thedilution effect on a signal from multiple expression vectors in anygiven cell.

Host Cells Displaying Peptides

In an exemplary embodiment, peptides, encoded by oligonucleotidescomprising an expression library, are displayed within the secretorypathways and extracellular surfaces of host cells into which theexpression library has been introduced. Host cells transfected withexpression library sequences will co-express the membrane protein ofinterest with peptide molecules, passing simultaneously through thesecretory pathway with the effector peptide, and being also tethered tothe cell surface along with the membrane protein. Thus, the expressedpeptides will be present with the membrane protein molecules duringprocessing and, therefore, these peptides will be available to interactwith the membrane protein of interest, the processing enzymes of themembrane protein, or other macromolecules that are involved inprocessing of the membrane protein. Further, during transience throughsecretory pathways, the library peptides will be expressed underphysiological conditions native to the processing of the membraneprotein. Similarly, where the extracellular environment of the hostcells is maintained under substantially physiological conditions,library peptides on the extracellular surface will also be expressedunder conditions that preserve or approximate those native to membraneprotein processing in the extracellular environment.

The peptides are typically displayed under substantially physiologicalconditions on the surface of host cells, such as mammalian cells. Eachhost cell can express on its surface hundreds and possibly thousands ofcopies of one or more library peptides, a majority of which aretypically available for binding to extracellular membrane protein targetmolecules. The peptides are typically present on the surface of a cellfor a sustained period of time.

In certain embodiments, the host cells expressing the peptide librariesare freshly prepared or live cells. In other embodiments, the peptidelibrary expressing cells can be fixed, such as in para-formaldehyde orother suitable fixative. Such fixed peptide library-expressing cellsoptionally can be stored at a suitable temperature (e.g., 4° C.) untiluse. The peptides are typically presented on the surface of the cellsfor a sustained period of time.

In some embodiments, host cells transfected with an expression libraryare enriched for cells that contain the expression vector and optimallyexpressing a library peptide. Such selection is typically based onselectable markers contained within the library expression vector (seeExpression Cassettes and Vectors). Methods for selection usingselectable markers are known in the art. For example, FACS can be useddetect fluorescently labeled antibodies to epitopes encoded by theexpression vector (e.g., V5, FLAG, thioredoxin, and the like) or to thepresentation molecule itself (e.g., CD24). In addition, magnetic beadselection can be used by known methods.

Prescreening of Agents for Agents That Specifically Bind to the MembraneProtein

In certain embodiments, compound libraries and expression libraries canbe prescreened to identify agents that specifically bind to the membraneprotein of interest. Prescreening can be performed, for example, undersubstantially physiological conditions. Agents identified asspecifically binding to the membrane protein of interest, or afunctional fragment thereof, can be used to generate a compound orexpression library enriched for membrane protein-binding agents(“pre-enriched compound library” or “pre-enriched expression library”).In one embodiment, enrichment can be accomplished by expressing theexternal domain of APP (edAPP) with an affinity tag (such as a His tag),binding the edAPP to a column, passing compound libraries over thecolumn, and eluting the enriched compounds from the column. The enrichedcompounds can then be tested on the host cells.

Further, in certain embodiments, N-terminal truncated forms of APP areused to prescreen agents for specific binding to APP. These truncatedforms have the advantage of being a more specific target for peptideenrichment. The utility of using the truncated forms is underscoredspecific target for peptide enrichment. The utility of using thetruncated forms is underscored by the fact that the α and β cleavagesites are not significantly affected by removal of most of theN-terminal sequence APP (see De Stooper et al., J. Biol. Chem.270:30310-30314, 1995; Lammich et al., Proc. Natl. Acad. Sci. USA96:3922-3927, 1999).

In an exemplary embodiment of the invention, the peptide expressionlibraries are prescreened to identify oligonucleotides encoding peptidesthat specifically bind to the membrane protein of interest or afunctional fragment thereof. Those clones identified as encodingmembrane protein-binding peptides can be recovered and amplified usingmethods known in the art to produce a pre-enriched expression library.This pre-enriched expression library comprises a population ofoligonucleotide sequences enriched for those that encode peptides thatbind to the membrane protein of interest. The enriched library ofoligonucleotide sequences can then be introduced into host cellsaccording to the methods of the present invention to identify thosemembrane protein-binding peptides that alter processing of the membraneprotein of interest.

For example, using methods known in the art, the expression library canbe introduced into an animal host cell for expression or, alternatively,expressed using a non-animal system such as, e.g., phage display. Theexpressed peptides can then be contacted with labeled membrane proteinand/or N-terminal truncated forms of the membrane protein of interest,either soluble forms (e.g., the extracellular domain of the membraneprotein or a N-terminal truncated form) or expressed on the surface ofanimal host cells. Suitable labels include, for example, radioactivelabels (e.g. ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I, ¹³¹I, and the like), fluorescentmolecules (e.g., fluoroscein isothiocyanate (FITC), rhodamine,phycoerythrin (PE), phycocyanin, allophycocyanin, ortho-phthaldehyde,fluorescamine, peridinin-chlorophyll a (PerCP), Cy3 (indocarbocyanine),Cy5 (indodicarbocyanine), lanthanide phosphors, and the like), enzymes(e.g., horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), biotinyl groups, tag epitopes as described above, and thelike. In some embodiments, detectable labels are attached by spacer armsof various lengths to reduce potential steric hindrance. Alternatively,labeled binding partners, such as, for example, antibodies that bind tothe target molecules can be used. In one exemplary embodiment, the APPmolecules are labeled with a His tag epitope label.

The expressed peptides that bind to the membrane protein of interest orone of the truncated forms thereof can then be identified using thelabel and methods known in the art. For example, phage or cellsexpressing peptides and exposed to His tag extracellular domain themembrane protein can be passed over a His tag affinity column to enrichfor those phage or cells expressing the membrane protein-bindingpeptides. Alternatively, for example, peptide-expressing animal cellscan be exposed to fluorescently tagged extracellular membrane protein ora truncated for the membrane protein and membrane protein-binding cellscan be identified and sorted by FACS to obtain cells expressing thepeptides that specifically bind to the membrane protein of interest.Multiple rounds of such enrichment can be performed.

After enrichment of cells or phage expressing membrane protein-bindingpeptides, the peptide encoding sequences can be excised from theexpression vector used for pre-enrichment and transferred, using knownmethods, to an appropriate vector (see Expression Cassettes and Vectors,infra) for screening for those membrane protein-binding peptides thatalter processing of the membrane protein. In some embodiments of theinvention, where animal host cells are used for pre-enrichment, theexpression vector used for the screening of peptides that alterprocessing of the membrane protein can be the same as used for peptideexpression during pre-screening.

Detection of Agents That Alter Processing of the Membrane Protein

In another aspect, the effect of agents within the compound orexpression libraries on membrane protein processing is assayed. The hostcells expressing the membrane protein of interest, e.g., TGF-α, TNF-α,APP, TNFR, and the like, and at least one processing enzyme, e.g., TGF-αsecretase, TNF-α secretase, α-secretase, β-secretase, or γ-secretase,and TNFR secretase respectively, are contacted with the agents and thehost cells are then assayed for an effect on the processing of membraneprotein expressed by the host cell. In some embodiments, the host cellsare contacted with the agents under substantially physiologicalconditions. In an exemplary embodiment, the effect on APP processing ofpeptides comprising a peptide expression library is assayed, theexpression library having been introduced into the host cells (seeGenetic Libraries, Expression Cassettes and Vectors, Nucleic AcidTransfer, and Host Cells Displaying Peptides, supra). In anotherembodiment, an extract from, for example, a plant, e.g., a peat extract,comprising an agent of the present invention is admixed with the hostcells for a sufficient time period to detect an effect.

Effects of agents within the libraries on membrane protein processingcan be detected by any suitable detection means, such as, for example,the use of markers that specifically bind to particular fragments of themembrane protein. In the specific example of APP the fragments can be,for example, APPs-α, APPs-β, APPs-γ. In the examples of TGF-α, TNF-α andTNFR marker that bind specifically to the soluble forms of the membraneproteins can be used. Using such markers, the relative presence orabsence of a particular membrane protein fragment that correlates withprocessing, in comparison to host cells that have not been contactedwith agents or that do not express library peptide sequences, can bedetermined. For example, membrane protein fragment specific bindingmarkers can be labeled with fluorescent tags and the host cells assessedfor the relative presence or absence of such marker by known methodssuch as, e.g., (FACS) analysis. In an exemplary embodiment, the markersare antibodies specific for particular epitopes of the membrane proteinor membrane protein fragments. Antibodies to particular APP fragments,including APPs-β (such as, for example, A3 or 1G7 specific to APPmidregion, Koo et al., J. Biol. Chem. 269:17386-17389, 1994), APPs-α(6E10 specific to the carboxy terminal end of APPs-α or the region ofAPP between the β and α cleavage sites, Pirttila et al., Neuron Sci.127:90-95, 1994, McLaurin et al., Nature Med. 8:1263-1269, 2002, and p3(4G8, Pirttila et al., Neurol Sci. 127:90-95, 1994, McLaurin et al.,Nature Med. 8:1263-1269, 2002).

In an exemplary embodiment, where the host cells express more than onemembrane protein processing enzyme, the ratio of detection signals of atleast two labeled markers specific for at least two different membraneprotein fragments can be determined. For example, altered APP processingcan be determined by detecting a change in this ratio in host cellsexpressing library sequences. In host cells expressing both α- andβ-secretase, antibodies specific for APPs-α and APPs-β can be labeledwith two different fluorescent tags (e.g., FITC and PE) and the presenceof bound, labeled antibody determined by FACS. Host cells expressinglibrary peptides can then be selected according to an increase inAPPs-β:APPs-α signal ratio, indicating a reduction in the β-secretaseprocessing.

In some embodiments, agents identified as having an effect on theprocessing of the membrane protein can be used to build a sub-libraryenriched for agents that affect processing. This sub-library can then becontacted with host cells in subsequent rounds of screening to identifyagents with the desired characteristics (see Characterization of LibraryConstituents, infra).

In an exemplary embodiment, host cells expressing a peptide library thatare isolated or collected by any of the methods described herein can beused to re-isolate the genetic library sequences(s) so as to build asub-library of sequences enriched for those that affect processing. Aswill be appreciated by those skilled in the art, such sequences can beisolated by, among other methods, recovering expression vector nucleicacids from the selected clones and transforming them into a suitablebacterial host strain, by cloning the library oligonucleotides by PCRusing any suitable priming site(s) that flanks the oligonucleotideinserts, by subcloning the oligonucleotides from the original expressionvector into another vector, and the like. The sub-library ofoligonucleotides optionally can be recloned into an expression cassetteor vector, as necessary, and reintroduced into the host cells forsubsequent rounds of screening. Screening/selection cycles can berepeated as many times as necessary.

In certain embodiments, after a sufficient number of cycles, asubstantial difference is observed in the assay signals or ratiosthereof, indicating changes in the presence of fragments of the membraneprotein between an enriched sub-library of peptide sequences and theoriginal peptide library (e.g., in an intensity distribution). By theprocess of sequential introduction of the expression library, orportions thereof, sorting in a flow sorter or similar device andisolation of nucleic acids from host cells exhibiting the desired assayresults, a population of library oligonucleotides can be identified thatencode the desired peptide(s). The oligonucleotides can then be isolatedand studied individually by molecular cloning and nucleic acid sequenceanalysis. If a sufficient number of cycles have been carried out, many,and typically most, separate oligonucleotides should encode a peptidethat produces about the same effect binding event when expressed on hostcells.

Identification of Allosteric Effectors

In certain embodiments, effector agents can be assayed to identifyallosteric effectors of the membrane protein of interest. Identificationof allosteric effectors can be performed as a secondary screen on agentsthat have been identified as effectors of membrane protein processing.Alternatively, compound libraries or peptide expression libraries can beprescreened to identify agents that bind to a potential allosteric siteon the membrane protein. Prescreens comprise detecting a conformationalchange in the membrane protein bound to the agent. The identification ofallosteric effectors can be done directly using a cellular assay whereeffectors of secretase, the membrane protein, e.g., APP (allosteric),and any other interaction that causes differential processing at any ofthe processing sites are identified. Those that cause differentialprocessing by binding to the membrane protein can be identified fromthis group. In addition, by prescreening for membrane protein bindingcompounds and then screening with the cell based assay, the system ispredisposed to the identification of allosteric effectors of themembrane protein.

Characterization of Library Constituents

Effector agents identified using any of the procedures described hereincan be further characterized. Where expression libraries are used todisplay peptides within secretory pathways and on the extracellularsurface of host cells, library sequences can be isolated from host cellsby any suitable method, such as, for example, HIRT lysis and recovery ofvectors in bacterial host cells, polymerase chain reaction, and thelike. (See, e.g., Hirt, J. Mol. Biol. 26:365-369, 1967; U.S. Pat. Nos.4,683,202, 4,683,195 and 4,800,159; Innis et al., PCR Protocols: A Guideto Methods and Applications, Academic Press, Inc., San Diego, Calif.(1989); Innis et al., PCR Applications: Protocols for FunctionalGenomics, Academic Press, Inc., San Diego, Calif. (1999); White (ed.),PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering,Humana Press, (1996); EP 320 308; Ausubel et al., supra; Sambrook etal., supra; which are incorporated by reference herein in theirentirety.)

Subsequent rounds of screening optionally can be performed to enrich foragents that alter processing of the membrane protein. Sub-libraries ofcompound or expression libraries can be passed through additionalscreens and/or selections to enrich for those agents or oligonucleotidesequences that have more desirable properties. To enrich for effectoragents that have more favored properties, it can be desirable to passagea sub-library (that has been isolated by any of the methods describedabove) through additional screens to enrich for those agents with, forexample, improved specificity or avidity for the membrane protein orwith an increased effect on processing of the membrane protein. Forinstance, minor effects on an undesirable secretory or extracellularinteraction can be eliminated by appropriate secondary screens. Ifdesired, additional labels can be used to identify library peptidesequences that affect secretory or extracellular molecular interactions.In addition, effector agents that have generalized, non-specific effectson secretory or extracellular interactions can be identified bycontacting compound sub-libraries or individual effector agents with (orpassing expression sub-libraries or individual library peptide sequencesthrough) different host cells that lack the membrane protein or membraneprotein processing enzyme expression and then conducting screens onthose cells that assay for processing alterations or other effects onother, non-membrane protein secretory or cell-surface molecules.

In some cases, effector agents identified according to the presentinvention can be used to identify other agents that alter processing ofthe membrane protein. For example, small organic molecules identified asmolecular effectors of membrane protein processing can used indirected-screening approaches to identify agents with improvedcharacteristics, including, e.g., reduced toxicity or increasedefficacy. For example, modifications can be made to one or more subunitscomprising the effector agent. Modifications can include variation as totype, number, or position of R groups. Modified small molecule effectoragents can then be screened using the methods described herein foragents with the improved characteristics.

Further, where peptide libraries are used, including peptide expressionlibraries, peptide or oligonucleotide sequences can be modified. In somecases, an original library may not contain all possible permutations ofan amino acid sequence of length N (e.g., when the original library is asemi-random library). In such cases, it can be possible to isolate anduse the identified peptides as a starting material (“lead compound”) toidentify additional peptides or peptides with enhanced function (e.g.,higher avidity or affinity) as compared with the original peptide. Toisolate variants of a library sequences, amplification of nucleic acids(e.g. by polymerase chain reaction) can be used to introduce sequencechanges during the replication process. (See, e.g., Cline et al.,Nucleic Acids Res. 24:3546-3551, 1996). Such mutations can lead tosequence variants that have more effective properties. Alternatively, itcan be desirable to seek improved variants of existing sequences bydeliberately subjecting the amplification process to conditions thatenhance mutation and/or recombination of the nucleic acid(s), such asby, for example, in vitro mutagenesis, error-prone PCR and/orrecombinational PCR. (See, e.g., Ausubel et al., supra; Stemmer, Nature370:389-391, 1994). Such conditions are known in the art and provide ameans for searching for sequences that are active at lowerconcentrations and/or that demonstrate increased specificity and/oractivity compared to the sequences expressed by the original library.

Applications of the Molecular Effectors of Membrane Protein Processing

The methods according to the present invention provide the ability toidentify physiologically relevant effector agents that bind and alterthe processing of the membrane protein, particularly under physiologicalconditions.

Effector agents identified using the methods described herein for APPprocessing can be tested in, e.g., a transgenic mouse model, such as,for example, Tg (HuAPP695.K670N/M671L) (Hasio et al., Science274:99-102, 1996) or the PDAPP (V171F) (Games et al., Nature373:523-527, 1995), to study their efficacy in plaque inhibition andcapacity to reduce Aβ levels in CSF, brain cells, and serum. Effectoragents can be administered by injection or orally. In addition, wherethe effector agent is a peptide, the identified peptide can be also betested by expressing the peptide in the mouse model. The, identifiedeffector peptides can also be expressed and presented extracellularly orexpressed and secreted in specific tissues. Other transgenic andnon-transgenic animal models are well known in the art for othersecreted proteins of the present invention that can be used to test theeffectors identified by the methods disclosed herein.

Additional Modifications to Enhance Function of Peptide Effectors

As discussed previously, peptide sequences that affect differentialprocessing of the membrane protein can exert their effect in a varietyof ways. As will be appreciated by those skilled in the art, it can bepossible to improve the effectiveness of a peptide by synthesizingpeptide variants or analogs. For example, the effectiveness of peptidesmight be improved by administering the peptides themselves (i.e.,without any extra sequences).

One skilled in the art will appreciate that structural analogs andderivatives of peptides (e.g., peptides having conservative amino acidinsertions, deletions or substitutions, peptidomimetics, disulphidecross-linking, artificial cross-linking, or the like) can also be usefulas therapeutic agents. For example, in addition to the above-describedpeptides, which can comprise naturally-occurring amino acids, peptideanalogs can be used as non-peptide drugs with properties analogous tothose of the template peptide. These types of non-peptide compounds canbe developed, for example, with the aid of computerized molecularmodeling. (See, e.g., Fauchere, Adv. Drug Res. 15:29, 1986; Veber andFreidinger, TINS 392, 1985; Evans et al., J. Med. Chem. 30:1229, 1987).Such analogs, or peptide mimetics, are structurally similar totherapeutically or prophylactically useful peptides and can be used toproduce an equivalent therapeutic or prophylactic effect. In some cases,peptide mimetics can have significant advantages over peptides,including, for example, more economical production, greater chemicalstability, enhanced pharmacological properties (e.g., half-life,absorption, potency, efficacy, and the like), altered specificity (e.g.,a broad-spectrum of biological activities), increased, reducedantigenicity, increased passage over the blood brain barrier, and otherdesired properties.

Peptide mimetics can be generated by methods known in the art andfurther described in the following references: Spatola, Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins (Weinstein, ed.)267, 1983; Spatola, Vega Data, Vol. 1, Issue 3, “Peptide BackboneModifications” (March 1983); Morley, Trends Pharm Sci., pp. 463-468,1980; Hudson et al., Int. J. Pept. Prot. Res. 14:177-185, 1979; Spatolaet al., Life Sci. 38:1243-1249, 1986; Hann, J. Chem. Soc. Perkin Trans.I, pp. 307-314, 1982; Alnquist et al., J Med. Chem. 23: 1392-1398, 1980;Jennings-White et al., Tetrahedron Lett. 23:2533, 1982; European PatentApplication EP 45665, 1982; Chemical Abstract 97:39405, 1982; Holladayet al., Tetrahedron Lett. 24:4401-4404, 1983; and Hruby, Life Sci.31:189-199, 1982.

In one aspect, pharmaceutically acceptable salts of a peptide (or analogor mimetic) can be readily prepared by conventional methods. Forexample, such a salt can be prepared by treating the peptide with anaqueous solution of the desired pharmaceutically acceptable metallichydroxide or other metallic base and then evaporating the resultingsolution to dryness, typically under reduced pressure in a nitrogenatmosphere. Alternatively, a solution of a peptide can be mixed with analkoxide of the desired metal, and the solution subsequently evaporatedto dryness. The pharmaceutically acceptable hydroxides, bases, andalkoxides encompass those with cations for this purpose, including, butnot limited to, potassium, sodium, ammonium, calcium, and magnesium.Other representative pharmaceutically acceptable salts includehydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate,valerate, oleate, laurate, borate, benzoate, lactate, phosphate,tosylate, citrate, maleate, fumarate, succinate, tartrate, and the like.

It can be desirable to stabilize the peptides or their analogs orderivatives to increase their shelf-life and pharmacokinetic half-life.Shelf-life stability can be improved by adding excipients such as: a)hydrophobic agents (e.g., glycerol); b) sugars (e.g., sucrose, mannose,sorbitol, rhamnose, or xylose); c) complex carbohydrates (e.g.,lactose); and/or d) bacteriostatic agents. The pharmacokinetic half-lifeof the peptides can be modified by coupling to carrier peptides,polypeptides, and carbohydrates using chemical derivatization (e.g., bycoupling side chain or N- or C-terminal residues), or by chemicallyaltering an amino acid of the subject peptide. The pharmacokinetichalf-life and pharmacodynamics of these peptides can also be modifiedby: a) encapsulation (e.g., in liposomes); b) controlling the degree ofhydration (e.g., by controlling the extent and type of glycosylation ofthe peptide); c) controlling the electrostatic charge and hydrophobicityof the peptide, and d) formulation in a pharmaceutically acceptabledepots such as polyactic acid, polyglycolic acid, polylactic-co-glycolic acid, or the like.

EXAMPLES

The following examples are provided merely as illustrative of variousaspects of the invention and should not be construed to limit theinvention in any way.

Example 1 Preparation of a Genetic Library in a Host Cell

A genetic library is prepared by inserting random oligonucleotides intoa cloning site of an expression vector. The expression vector has anexpression cassette comprising, in a 5′ direction relative to thedirection of transcription, a promoter, a nucleic acid encoding a signalsequence, a nucleic acid encoding a presentation molecule, a cloningsite located at the 5′ end of the nucleic acid encoding the presentationmolecule, a nucleic acid encoding a transmembrane domain, and atranscription terminator. The expression vector includes an origin ofreplication (ColE1) and an antibiotic resistance marker for selection inE. coli. The random oligonucleotides encode peptides of about 7 to about20 amino acid residues. The vectors containing the oligonucleotides aretransformed into host bacteria and grown under selectable conditions toestablish a library of about 10 million to several billion independentisolates. Vector DNA is prepared from this library. This vector DNA isintroduced into animal cells, such as, for example, human cells,mammalian cells, or other animal cells.

Example 2 Engineering the Random Peptide Vector for the Expression ofAPP Effectors

The preferred random peptide vector for the expression of APP effectorsis a retroviral vector with the cassette insert shown in the FIG. 3. Thecassette encodes a promoter; a secretory sequence to cause the proteinto enter the secretory pathway; a random peptide sequence encodingcysteines at the termini of the random sequence to cause disulfidebridge formation lending structure to the random amino acid sequence; aglycine spacer; a presentation protein; a second glycine spacer toimpart flexibility at the cell surface; and a GPI linker sequence thatcauses the fusion protein to be tethered to the cell surface. Thepresentation protein is a globular inert protein on which the randompeptide sequence is tethered and displayed. This configuration allowsthe peptide ring of random amino acids to be tethered at the end of astring of glycines providing flexibility. The glycine spacer between thecell and the presentation protein also allows for flexibility of thewhole tethered molecule. Flexibility for the peptide ring minimizessteric hindrance from the presentation protein with the binding of therandom amino acid sequence to APP.

Example 3 Expression Vector Construction

To achieve high expression levels of library peptides on the surface ofhost cells, an expression vector is used. The expression vector includesmarkers required for propagation and selection in bacteria, anexpression cassette including a mammalian cell transcription promoter(e.g., the cytomegalovirus or EF-1α promoter, and the like), a nucleicacid encoding a presentation molecule and a transcription terminator.Random library sequences can be inserted at the N-terminus, at theC-terminus, or internally in the nucleic acid encoding the presentationmolecule and can be in a linear or constrained loop array or in anexposed loop of the presentation molecule.

To attach the presentation molecule to the surface of host cells, thenucleic acid encoding the presentation molecule includes a sequenceencoding a secretory signal sequence and an element to tether the fusionprotein to the cell surface (e.g., a signal forglycophosphatidylinositol (GPI) anchorage or a transmembrane andintracellular domain). Suitable presentation molecules include, forexample, the IL-3 receptor, protein A thioredoxin, CD4, CD20, or CD24.The presentation molecule can also include one or more epitopes, suchas, for example, FLAG, V5 or polyhistidine. The transcriptionterminator, can be, for example, from human growth hormone.

Two distinct expression vectors were constructed to display peptidelibraries on the surface of cells of mammalian cells such as COS7 andK562 cells. One construct places the peptides, having 7 amino acidresidues, at the N-terminus as a linear structure, while the otherconstruct includes a cysteine residue at each end of the peptidesequences to form a constrained loop at the N-terminus. Each constructencodes a presentation molecule including thioredoxin, the V5 or FLAGepitopes, the secretory signal sequence from CD24 for secretion, and theGPI linkage sequence from CD24 for attachment to the surface of the hostcell. The approximate diversity of each of the completed peptideexpression vectors is about 1×10⁹ unique peptides, although libraries ofconsiderably greater diversity can be produced.

Example 4 Establishment of Assay Cell Lines for the Identification ofAPP Effectors

There are four primary requirements for the assay cell line to identifyeffectors of APP: 1) the cells must possess the natural complement ofAPP processing enzymes, 2) the cells must constitutively produce APP, 3)the cells are suspension cells to facilitate the high throughputrequirement of these experiments, and 4) the screening system needs tomimic the natural physiological conditions associated with APPexpression.

APP expressing cell lines are prepared by constructing a mammalianexpression vector encoding APP and introducing this expression vectorinto the parent cell line. Two cell lines, a neuroblastoma cell line anda human embryonic kidney cell line (HEK), are preferred for theseexperimentations. Both of these cell lines have been used in APPexperimentation (Cedazo-Minguez et al., Neurochem. Int. 35:307-315,1999; Lopez-Perez et al., J. Neurochem. 73:2056-2062, 1999). The HEKcell line is easier to grow and presents fewer challenges than theneuroblastoma cell line and therefore is preferred for theseexperiments. The neuroblastoma cell line is an alternative cell line.

To facilitate these experiments, the HEK cells are adapted to spinnerculture conditions. Adaptation is accomplished by gradually reducing theamount of serum in the culture medium in static cultures until the cellsare capable of growing in serum free media. The cells are thentransferred and grown in spinner culture vessels. There is significantcell death with both of these procedures but some cells survive andgrow. The cells that grow the best under these conditions outgrow cellsthat grow less well, producing cells adapted to grow well under serumfree spinner culture conditions. These procedures will be understood bysomeone skilled in this art.

The cDNA encoding APP may be obtained from ATCC or IMAT or alternativelyobtained by PCR amplification and verified by DNA sequence analysis. TheAPP encoding cDNA is inserted into a mammalian expression vector andelectroporated into the parental mammalian cell line. The electroporatedcells are plated in microtiter plates at a density to allow for cellgrowth in 50% of the wells under neomycin selection, to provideconditions allowing for clonal isolation. These cell lines are assessedfor APP expression by fluorescent microscopy and FACS analysis usinganti-APP antibodies as described below.

To successfully implement a screening strategy as described in thisapplication, it is important to be able to manipulate and screen largenumbers of cells and peptide sequences. By utilizing suspension culturecells and spinner culture vessels, methods of growing, handling, andutilizing billions of cells per day can be implemented.

Example 5 In Vitro Expression of APP and Analysis of Products

Clones of wild type APP and APP containing the Swedish, Arctic, and/orDutch mutations were expressed in HEK-293 cells. Expression of themutant clones are useful as controls to show differences in processingin an in vitro screening assay screening assay for effectors of APPprocessing.

To construct the expression plasmids, a clone encoding the full-lengthwild type 695 amino acid human APP (Kang et al., Nature, 325:733-736,1987) was obtained. A 3 kb fragment from the NruI site in exon 1 to theSmaI in the 3′UTR was subcloned into the pcDNA3.1 vector. This vectorcontains a CMV promoter and an SV40 polyadenylation sequence. Similarly,clones encoding APP_(Swe) and APP_(Arc) were used as starting materialfor the construction of CMV-APP_(Swe) and CMV-APP_(SweArc). Theseconstructs were introduced into HEK-293 cells. Cleavage by endogenoussecretases results in processing of the APP protein into severalfragments. APP protein expression and processing was assessed by Westernblot analysis of lysates and conditioned media of mock-transfected(i.e., transfected with a plasmid lacking the APP sequence) andAPP_(Swe)-transfected cells. Full-length APP as well as C-terminalfragment C99, Aβ, APP-α, and APPs-β were detected using 6E10 antibody,which recognizes amino acids 1-16 of Aβ sequence. Full-length APP, C99,and Aβ were found in cell lysates from APP_(Swe) transfectants. Theidentification of Aβ in the cell lysate is believed to be the result ofprocessing within the secretory pathway. APPs-α and Aβ were detected inconditioned medium from APP_(Swe) transfectants. APPs-α and APPs-β werealso detected in conditioned medium of cells transfected with theSwedish or Swedish/Arctic mutation using 6E10 antibody and Sw192antibody, respectively. Antibody Sw192 (Elan Pharmeceuticals) recognizesthe amino acids 590-596 of APP_(Swe) only when it has been cleaved byβ-secretase.

Example 6 Retroviral Infection

The retroviral expression vector is packaged in a packing cell line(Miller et al., Methods Enzymol. 217:581-599, 1993) and utilized toinfect the assay cells containing secretase processing enzymes and APP.The fusion protein will enter and pass through the secretory pathwayunder the influence of the secretory signal concluding with presentationon the cell surface. At the cell surface, the fusion protein comprisingthe presentation protein, glycine spacers and random amino acid sequence(display complex) becomes attached to the cell via the GPI linkage.Processing of APP by some of the secretases appears to occur duringtransience through the secretory pathway as well as at the cell surface(Selkoe, Physiolgical Rev. 81:741-767, 2001). The display complex andAPP will similarly pass through the secretory pathway and be displayedon the surface of the cell in a common process. Therefore, the randomamino acid sequence will be present and the effective peptide will havethe opportunity to bind to APP altering its structure within thesecretory pathway as well as at the cell surface. FIG. 4 depicts theconfiguration of the display complex.

Example 7 Enrichment of Transfected Cells

Cells vary in the efficiency to take up DNA and express it. In caseswhere introduction of expressible DNA is not very efficient, it canoptionally be possible to enrich for cells that contain the expressionvector (e.g., plasmid) and are optimally expressing the DNA. To allowenrichment of cells that contain the genetic library, a label, such as asequence encoding a marker, is included in the expression vector. Such amarker typically will remain attached to the cell. This sequenceencoding the marker can be, for example, a transcription promoter andterminator, a sequence encoding a secretory signal sequence (e.g.,CD24), one or more extracellular domains (e.g., V5, FLAG, and the like)and a sequence to tether the marker to the cell surface (e.g., a signalfor glycophosphatidylinositol (GPI) anchorage). The placement of peptidelibraries between two distinct markers (e.g., V5 and FLAG) can ensurethe integrity of the peptide library by selecting only cells thatcontain both markers in the presentation molecule and that are expressedon the surface of host cells.

The expression vectors described in Example 3 have been used totransfect COS7 cells by electroporation employing conditions tointroduce, on average, only one or a few vectors per cell. The cellswere placed in culture following transfection and analyzed at varioustimes by FACS to detect the expression of thioredoxin or FLAG on thesurface or interior of cells. In each case, a relatively high percentageof cells express the presentation molecules (e.g., thioredoxin-detectedusing anti-thioredoxin antibody) one day post-transfection, and themolecules persisted over the next week. These results indicate thatpresentation molecules are expressed at relatively high levels overseveral days.

Transfected host cell, expressing a peptide library were also fixed inpara-formaldehyde prior to FACS analysis or selection on magnetic beads.The results demonstrated that the marker epitopes were still accessibleto antibody following fixation, indicating that the library peptideswere available for binding to target molecules.

Similar experiments demonstrated that K562 cells, transfected byelectroporation with plasmid vectors encoding a peptide library, weretransfected at approximately 50% efficiency with 80% cell survival. Theoptimum expression period was between one and two days followingtransfection. The level of expressed presentation molecule on K562 cellswas lower than that on COS7 cells, because K562 cells do not amplify theplasmid vectors as do COS7 cells. However, sufficient presentationmolecules were expressed on the surface of K562 cells as demonstrated bylocalization of labeled anti-FLAG antibody.

The results demonstrate a robust system for displaying peptide librarieson the surface of mammalian cells. The tethering of the presentationmolecule via a GPI linkage to the cell and the use of domains fromthioredoxin and CD24 lead to the persistent, stable expression ofpeptide libraries. Further, the placement of the peptides at theN-terminus of the presentation molecule ensures unobstructedaccessibility of the peptides to potential target molecules, relativelydistant from the cell surface and in a highly favorable hydrophilicenvironment.

Example 8 Prescreening for Peptides that Bind APP

Because of the interest in peptide structures that bind and alter thestructure of APP, and because not every peptide that binds to APP willmodulate proteolytic processing of the protein, however, all peptidesthat affect processing can be expected to have a high affinity bindingfor APP, a pre-enrichment of peptides that simply bind APP is performed.Phage display is used for this enrichment process because of the largenumber of structures that may be generated and screened by this method.The APP extracellular domain and two N-terminal truncated forms, onebeginning at nucleotide 457 just 3′ to the Kunitz protease inhibitordomain sequence and the second at nucleotide 550 are expressed inbacteria and CHO-DG44 cells with a His tag for easy purification. Theproduction, stability, and ease of purification of these affinitymolecules determine the affinity molecule of choice.

Phage expressing random peptide sequence are exposed to His tagextracellular domain APPs and passed over a His affinity column. After 2or 3 rounds of enrichment the nucleotide sequences encoding the randompeptides are excised from the DNA of enriched phage and transferred tothe retroviral vector. This enrichment step increases the probability ofidentification of a peptide that alters APP processing by binding to APPrather than altering the activity of the processing enzymes or causing achange in processing in some other manner.

Example 9 Screening for Effector Peptides

Assay cells are grown in large numbers, approximately a billion or more.The cells are infected with retrovirus encoding a peptide library. Aftertwo days of growth, the cells are selected by magnetic bead selectionfor those cells expressing the display complex utilizing biotinylatedanti-presentation protein antibody and strepavidin coated beads(Miltenyi Biotec, Germany). Selection enriches for cells expressing thedisplay complex on the surface of cells. This eliminates cells that arenot infected and cells expressing a sequence that is prematurelyterminated by a stop codon in the random DNA sequence. Semi-random DNAencoding the random peptide sequence is used to minimize stop codons butdoes not completely eliminate it (LaBean and Kauffman, Protein Sci.2:1249-1254, 1993). The frequency of stop codons is related to thelength of the random peptide sequence. An amino acid length of about 7to about 12, or about 16 will typically be used.

Magnetic bead selection also eliminates cells that have been infected,have the capacity to synthesize full length tethered display complex,but do not express sufficient amounts of the display complex due to, forexample, integration into a silent expression site in the chromosome.Selection therefore provides cells that are expressing sufficient levelsof display protein to be effective in altering the structure of APP. Thevalidity of this configuration has been demonstrated by expressingtethered effector peptide sequence in growth dependent Ba-F3 cellsexpressing thrombopoietin receptor. The tethered effector peptideconfers growth independence to the previously growth dependent cellline. This demonstrates that a tethered peptide can alter the structureof a cell surface receptor causing activation of the receptor and cellsurvival.

Bead enriched cells are sorted by fluorescent activated cell sorting(FACS) utilizing differentially labeled antibodies recognizing differentfragments of APP. To enrich for peptides causing increased α-secretaseprocessing, sorting will be performed based on a change in the ratio offluorescence between PE labeled antibody (6E10, Pirttila et al., NeurolSci. 127:90-95, 1994, McLaurin et al., Nat. Med. 8:1263-1269, 2002)recognizing amino acid 3-10 in Aβ corresponding to the C-terminal end ofAPPs-α and FITC labeled anti-p3 antibody (4G8, Pirttila et al., NeurolSci. 127:90-95, 1994, McLaurin et al., Nature Med 8:1263-1269, 2002)recognizing amino acid 16-24 of Aβ, corresponding to the N-terminal endof p3 (See FIG. 5). Control cells infected with the expression vectorwithout random peptide sequence provide a control PE/FITC ratio. Cellswith a lowered PE/FITC fluorescent ratio are collected.

Similarly, cells are sorted for peptides causing a reduction inβ-secretase processing by collecting cells with increased ratio of FITClabeled anti-APP-β. (A3 or 1 G7 specific to APP midregion, Koo et al.,J. Biol. Chem. 269:17386-17389, 1994) to PE labeled antibody (6E10)anti-APPs-α fluorescence (See FIG. 5).

To set up and test the APP effector assay system, reagents andconditions are used that influence the amount of cleavage at the α and βsites to demonstrate the sensitivity and validity of the assay. Forexample, assay cells are transfected with expression vectors encodingeither α or β-secretase. This increases processing at these sites and isrevealed by the cellular assay. Once enriched cells are obtained byFACS, the DNA encoding the random peptide is recovered from the cells byPCR amplification and recloned into the peptide library expressionvector. These are procedures known to someone trained in this art. Theisolated clones are amplified in bacteria, repackaged in the retroviralpacking cells, used to infect naive assay cells, and the enrichmentprocess is repeated until clones encoding true effector peptides areobtained. DNA sequence analysis of the clones encoding the randompeptide sequence reveals the sequence of the effector peptide.

Once one or more effector peptides have been identified, the effectorpeptides can be tested for their ability to effect the structure of APP.Methods for testing or analyzing the binding of the effector peptidesare well known and include methods described above for prescreeningpeptide libraries.

Example 10 Characterization of Effector Peptide

The effector peptide is characterized to show that soluble Aβ is reducedin cultures under the influence of the effector peptide. ELISA andWestern blot analysis of culture medium from assay cells plus and minusexpression of effector peptide can be used to verify the desired effect.

In one assay, an effector peptide that increase α-secretase processingof APP (herein “EMα peptide”) is coexpressed with human APP in HEK293cells or SH-SY5Y cells. The expression level of full length APP isdetermined in the presence or absence of the effector peptide by Westernblot analysis (antibody 6E10 or 22C11). Furthermore, efficacy testing ofthe effector peptide assesses its capacity to reduce protofibrils andmonomeric soluble Aβ1-40 and Aβ1-42 levels in the media after peptideexpressi`on. Levels of Aβ protofibrils and Aβ1-40/42 is determined byELISA, using different antibodies. The commercially available antibodies(i.e., Biosource/QCB cat # 44-348 and 44-344) are used to specificallyquantitate Aβ40 and Aβ42, respectively.

Similarly, the effect of peptides that decrease β-secretase processingof APP (herein “EMβ peptide”) on the expression level of APP and Aβ40and Aβ42 is characterized in a similar manner.

The effect of EMα and EMβ peptides on nearby processing activities isalso characterized. In cells expressing an EMα peptide, the release ofAPPsβ, APPsγ and as well as the C-terminal fragments CT99, is comparedto the release of these fragments when the EMα peptide is not expressed.A different fragment pattern indicates interference. In analogy, therelease of APPsα, APPsγ, p3, and CT 57/59 is determined with and withoutexpression of the EMβ peptide.

Whether the specificity or efficacy of an EMα or EMβ peptide is alteredin familial AD mutations is also determined. The following APP mutationsare used for this study:

-   -   (a) Swedish (Lys670Asn; Met671Leu), 2 amino acids N-terminally        to the β-cleavage site;    -   (b) Flemish (Ala692Gly), 5 amino acids C-terminally to the        α-cleavage site;    -   (c) Dutch (Glu693Gln), 6 amino acids C-terminally to the        α-cleavage site;    -   (d) Arctic (Glu693Gly), 6 amino acids C-terminally to the        α-cleavage site; and    -   (e) Iowa (Asp694Asn), 7 amino acids C-terminally to the        α-cleavage site.

Also assessed is whether an APP mutation affects the affinity orassociation kinetics of an EMα or EMβ peptide to the APP protein and APPprocessing rates. Binding studies are performed by incubating aradiolabelled EMα or EMβ peptide with HEK293 cells expressing wild-typeAPP or mutated forms of APP.

Processing at the β-site is significantly increased when APP containsthe Swedish mutation. (See Mullan et al., Nat. Genet. 1:345-347, 1992.)For example, in transgenic mice expressing human APP with the Swedishmutation (hamster prion promoter-hAPP_(Swe)), SDS soluble Aβ40 and Aβ42peptides are increased from 21 and 6 pmol/g at 5 months of age to 11,100and 2,000 pmol/g at 21 months, respectively. (Kawarabayashi et al., J.Neurosci. 15:372-381, 2001). Co-transfection of an EMβ peptide withAPP_(Swe) indicates whether the Swedish mutation affects the EMβpeptide's inhibitory action on the β-site cleavage or not. Similarly,also assessed are the effects of APP mutations (b)-(e) near the α-site,see supra, on EMα peptide-stimulated α-cleavage.

The efficacy of an effector peptide (EP) in reducing the amount of Aβcan also be assessed in a suitable transgenic animal model that overexpresses Aβ, for example Tg(HuAPP695.K670N/M671L)2567 or PDAPP. (Seealso, e.g., Examples 11 and 12, infra.)

Additional characterization involves identification of the target of thepeptide. These assays utilize the display complex (presentation proteinwith tethered peptide) as well as the peptide alone as affinity labels.Binding of the peptide to APP is assessed by labeling the peptide andassessing binding to the extracellular domain of APP. Fluorescentlabeled peptide is also used to assess binding to the assay cellsexpressing APP, with a comparison to control assay cells not expressingAPP.

To assess binding to processing enzymes, labeled peptide is used todetermine peptide binding to parental assay cells and comparing peptidebinding to cells known to not express the processing enzymes. Additionalcharacterization can comprise labeled peptide mixed with solubilizedcell lysates and fractionated by ion exchange and size exclusionchromatography, 2-D electrophoresis, and mass spectrum analysis ofprotein spots to determine binding to other cell components. It ispossible that an effector peptide binds and alters the function ofanother cellular molecule causing a reduction in Aβ without binding toAPP or without altering the processing of other important biologicalmolecules.

Example 11 In Vivo Efficacy Testing of Effectors of APP Processing inTransgenic Mice—Direct Peptide Administration

In vivo efficacy testing of an EMα peptide will assess its capacity tostimulate APPα-secretase cleavage rates and lowering of Aβ protofibrilsand Aβ40/42 levels in the brain. Protofibril levels and braindistribution are assayed by ELISA and immunohistochemistry using aprotofibril-specific monoclonal antibody. Plaque burden is determinedusing standard procedures involving perfusion and fixation of braintissue in 4% paraformaldehyde and immunostaining with Aβ-antibody (e.g.,with 6E10 as previously described (see Nilsson et al., J. Neurosci.,21:1444-1451 2001)).

Initial testing of EMα and EMβ peptides involves direct administrationof the peptide by subcutaneous or intraperitoneal injections to Tg micefor measuring blood-brain-barrier passage properties. Passage isassessed by determination of the EMα or EMβ peptide in CSF and brainhomogenate after brain perfusion using an anti-EMα or anti-EMβ peptideELISA method, respectively. Different dose levels are tested andcorrelated to peptide levels obtainable in brain and CSF. In vitroexperiments provide information on the concentrations of the EMα or EMβpeptide required to obtain significant stimulation of α-secretase orinhibition of β-secretase, respectively. An alternative and moresensitive approach is to radiolabel (iodinate) the peptides anddetermine their radioactivity in CSF and brain homogenate. Although thismethod is more sensitive, it can give erroneous results due tomodification of the peptide. Furthermore, half-life for the EMα or EMβpeptide is determined in CSF, brain, and serum using standardprocedures, as a guidance for dosing frequency in the animal efficacystudies. A constant CSF/brain concentration of the EMα or EMβ peptideover time is typically desired. If the EMα or EMβ peptide showssatisfactory passage properties, then efficacy testing is performed.However, if passage properties of the EMα or EMβ peptide are notsatisfactory, then efficacy testing by direct subcutaneous orintraperitoneal. administration of the peptides is not be performed. Analternative approach is to deliver the peptides by i.c.v. using anosmotic pump (Alzet) or to directly express the peptide in the brain ofTg mice (see infra).

In one study, efficacy testing of the EMα or EMβ peptide is by directpeptide administration. Efficacy testing by direct administrationsinvolves administration of the EMα or EMβ peptide for 4-6 months to Tgmice, with reduction of Aβ40/42 and Aβ protofibril levels as anendpoint. A reduction of Aβ levels likely leads to a reduction of Aβprotofibrils, since formation of protofibrils are dependent on Aβ1-40and Aβ1-42 concentrations. A suitable mouse strain is mThy1-hAPP_(Swe),which carries a transgene coding for the 695-amino acid isoform of humanAmyloid Precursor Protein (APP) containing the Swedish mutation which isan ongoing project in our laboratory and similar to a well-establishedAD model (see Hsiao et al., Science 274:99-102, 1996). Thus, themThy1-hAPP_(Swe) likely shows a significant time-dependent increase ofAβ1-40 and Aβ1-42, large amyloid depositions, aged-correlated elevationof brain cholesterol and ApoE, as well as altered synaptic efficacy (seeKawarabayashi et al, J. Neurosci. 15:372-381, 2001). This “Hsiao-APPmouse” model has also been shown to develop behavioral deficits (seeWesterman et al., J. Neurosci. 22:1858-1867, 2002).

Efficacy testing is also performed in a double transgenic model(mThy1-hAPP_(SweArc)), containing both the Swedish and Arctic mutation.This model is developed to establish a model that produces high levelsof AβArc protofibrils in the brain. Aβ protofibrils are neurotoxic (seeHartley et al., J. Neurosci. 19:8876-8884, 1999) and affects earlysynaptic function (see Selkoe, Science 298:789-791, 2002). Nilsberth etal. (Nat. Neurosci. 4:887-893, 2001) has shown that the Arctic mutationconfers higher protofibril stability and rate of formation, leading toearly onset of AD.

Short-term administration (2-3 weeks) of the EMα or EMβ peptide at anearly age, prior to any amyloid deposition and when the Aβ levels showslittle age-dependent increase, assesses their mechanistic ability toalter APP processing in vivo. Further, the prophylactic effect of theEMα or EMβ peptide is evaluated in young Tg mice by long-termadministration starting prior to the onset of amyloid deposition andcontinuing until a time point when the transgenic mouse model is knownto display robust amyloid deposition. Finally, the therapeutic efficacyof the EMα or EMβ peptide is examined by long-term administration to oldmice after the onset of amyloid deposition. Various measures of amyloidpathology are determined to assess the efficacy and safety (e.g., Aβ andThioflavine S plaque burden, extractable Aβ1-40 and Aβ1-42, Aβprotofibrils as assessed by sequential extraction (TBS, Triton X-100,SDS and formic acid), and ELISA). Secondary tissue damage such asneurodegenerative changes (neuritic dystrophy, synaptic loss, oxidativedamage) is also analyzed preferentially with immunohistochemistry andquantitative image analysis. Traditional markers of cerebralinflammation such as GFAP (astrogliosis) and MAC-1/CD11 and IL-1(microgliosis) are determined, as well as other pro-inflammatorycytokines such as γ-IFN, IL-2, and IL-6 and anti-inflammatory cytokinessuch as TGF-α, IL-4, and IL-10. Prevention of cognitive dysfunction isstudied in the Radial Arm Water Maze, which is more sensitive to“episodic-like” memory than the classical Morris Water Maze.

Example 12 In Vivo Efficacy Testing of Effectors of APP Processing inTransgenic Mice—Peptide Expression In Vivo

If the blood-brain-barrier passage properties of the EMα or EMβ peptideare not satisfactory, an alternative strategy for efficacy testing isperformed by direct in vivo expression of the effector peptide in brainsof transgenic mice. Two different expression vector constructs aregenerated, both in which an EMα or EMβ peptide coding sequence isexpressed in neurons using, e.g., the Thy-1 promoter. One DNA construct(herein Thy 1-Emα/EMβ_(S)) is engineered so that the effector peptide isexpressed in a secreted (S) form in the brain; the other DNA construct(herein Thy1-Emα/EMβ_(M)) contains a GPI-linker and thereby targets theexpressed peptide to neuronal membranes (M). (See FIG. 8.)Thy1-Emα/EMβ_(S) and Thy1-Emα/EMβ_(M) transgenic mice are then generatedusing standard procedures.

Thy1-Emα/EMβ_(S) and Thy1-Emα/EMβ_(M) transgenic mice are crossed withThy1-hAPP_(Swe) and mThy1-hAPP_(SweArc) transgenic mice to determine theefficacy of the EMα or EMβ peptide in vivo. These multiple transgenicmodels are also used to characterized effect of the peptide oncompartmentalization of APP processing. The effect of secreted ormembrane-bound (e.g., ER-, Golgi-, plasma membrane-bound) EMα peptide onAPPα-secretase cleavage rates are determined. Similarly, the effect ofsecreted or membrane-bound EMβ peptide on β-secretase cleavage rates arealso examined. These studies provide important in vivo data, such as,e.g., whether increased α-site processing or deceased β-site processingtranslates into a reduction in Aβ protofibril and Aβ40/42 levels, lessamyloid depositions, less inflammation and/or improved cognitivefunctions.

The previous examples are provided to illustrate, but not to limit, thescope of the claimed inventions. Other variants of the inventions willbe readily apparent to those of ordinary skill in the art andencompassed by the appended claims. All publications, patents, patentapplications and other references cited herein and are also incorporatedby reference herein in their entirety.

1. A method for identifying an agent that alters processing of amembrane protein of interest, comprising: contacting the agent with ananimal host cell that expresses the membrane protein and at least oneprocessing enzyme of the membrane protein, and detecting alteredprocessing of the membrane protein to identify the agent that alters theprocessing of the membrane protein
 2. The method of claim 1, whereindetecting the altered membrane protein processing comprises assessingthe relative presence or absence of at least one species of membraneprotein fragment on the surface of the host cell or released from thesurface of the host cell.
 3. The method of claim 2, wherein the at leastone species of membrane protein fragment is released from the surface ofthe host cell.
 4. The method of claim 1, wherein the at least onemembrane protein processing enzyme is a protease or a phospholipase. 5.The method of claim 1, wherein the altered membrane protein processingresults in a decreased production of a fragment of the membrane proteinreleased from the cell surface.
 6. The method of claim 5, wherein thereleased fragment is associated with an increased risk of disease. 7.The method of claim 6, wherein the disease is an inflammatory disease,cancer, diabetes, Alzheimer's disease, or Parkinson's disease.
 8. Themethod of claim 1, wherein the agent is from a compound library.
 9. Themethod of claim 8, wherein the compound library is a combinatorialchemical library.
 10. The method of claim 8, wherein the compoundlibrary is a natural products library.
 11. The method of claim 8,wherein the compound library is a peptide library.
 12. The method ofclaim 1, wherein the agent is a small molecule.
 13. The method of claim1, wherein the agent is a biomolecule.
 14. The method of claim 13,wherein the biomolecule is a peptide.
 15. The method of claim 14,wherein the peptide is produced by transcription and translation from anoligonucleotide encoding the peptide.
 16. The method of claim 15,wherein the oligonucleotide has a length of about 18 to about 120nucleotides.
 17. The method of claim 15, wherein the oligonucleotide hasa length of about 36 to about 60 nucleotides.
 18. The method of claim15, wherein contacting the peptide with the host cell comprisesintroducing an expression vector, the expression vector comprising theoligonucleotide encoding the peptide, into the host cell, the host cellthereby expressing and displaying the peptide within a secretory pathwayand on an extracellular cell surface.
 19. The method of claim 15,wherein the oligonucleotide is from an expression library comprising aplurality of oligonucleotides, at least of majority of theoligonucleotides having different sequences encoding a differentpeptide.
 20. The method of claim 19, wherein the sequence of theplurality of oligonucleotides is randomized.
 21. The method of claim 19,wherein the expression library is pre-enriched for oligonucleotidesencoding peptides that specifically bind to the membrane protein. 22.The method of claim 19, wherein the contacting of the peptide with thehost cell comprises introducing the expression library into a firstplurality of animal host cells that express the membrane protein and atleast one processing enzyme of the membrane protein, the host cellsthereby expressing and displaying the different peptides within asecretory pathway and on an extracellular cell surface.
 23. The methodof claim 22, further comprising: selecting from the first plurality ofhost cells displaying the different peptides a first subset of hostcells that exhibit altered membrane protein processing; and identifyingfrom the first subset of host cells a first sub-library of theexpression library comprising at least one oligonucleotide that encodesthe peptide that alters the processing of the membrane protein.
 24. Themethod of claim 22, wherein the first plurality of host cells displayingthe different peptides have been enriched using a selectable marker. 25.The method of claim 24, wherein the selectable marker is V5, FLAG, orthioredoxin.
 26. The method of claim 24, wherein the enrichmentcomprises magnetic bead selection.
 27. The method of claim 24, whereinthe enrichment comprises selection by fluorescence-activated cellsorting.
 28. The method of claim 24, wherein the host cells expressingand displaying the different peptides on the extracellular cell surfaceexpress a high copy number of the different peptides.
 29. The method ofclaim 15, wherein the peptide is displayed as a fusion protein with apresentation molecule.
 30. The method of claim 29, wherein thepresentation molecule is CD24.
 31. The method of claim 29, wherein thepresentation molecule is IL-3 receptor.
 32. The method of claim 29,wherein the presentation molecule is thioredoxin.
 33. The method ofclaim 29, wherein the fusion protein further comprises a marker epitope.34. The method of claim 33, wherein the marker epitope is polyhistidine,V5, FLAG, or myc
 35. The method of claim 29, wherein the fusion proteinfurther includes a signal for a glycophosphatidylinositol (GPI)anchorage.
 36. The method of claim 1, wherein the animal host cell is amammalian host cell.
 37. The method of claim 1, wherein the animal hostcell is a recombinant host cell.
 38. The method of claim 37, wherein theanimal host cell is an isolated cell.
 39. The method of claim 1, whereinthe agent is contacted with the host cell under substantiallyphysiological conditions.
 40. The method of claim 39, wherein thesubstantially physiological conditions comprise the presence of acomplex biological fluid.
 41. The method of claim 40, wherein thecomplex biological fluid is blood, serum, plasma, or cerebral spinalfluid (CSF).
 42. The method of claim 2, wherein the assessment of therelative presence or absence of at least one species of a membraneprotein fragment on the cell surface comprises contacting the host cellwith at least one detectably labeled marker that specifically binds tothe at least one species of membrane fragment and detecting the bound,labeled marker.
 43. The method of claim 42, wherein the at least onemarker is an antibody that binds to a predetermined epitope of themembrane protein or a membrane protein fragment.
 44. The method of claim43, wherein the assessment of the relative presence or absence of atleast one species of membrane protein fragment on the cell surfacefurther comprises determining a ratio of the detection signals of atleast two labeled antibodies specific for at least two differentepitopes of the membrane protein or a membrane protein fragment.
 45. Themethod of claim 1, wherein the agent is an allosteric effector of themembrane protein.
 46. The method of claim 1, wherein the detectingaltered processing of the membrane protein on the surface of the hostcell comprises the use of a flow sorter.
 47. A method for identifying apeptide that alters processing of an angiotensin converting enzyme (ACE)membrane protein comprising: introducing an expression librarycomprising a plurality of oligonucleotides, at least a majority of theoligonucleotides having different sequences encoding different peptides,into a first plurality of animal host cells that express ACE membraneprotein and at least one ACE processing enzyme, the host cells therebyexpressing and displaying the different peptides within a secretorypathway and on an extracellular cell surface; selecting from the firstplurality of host cells displaying the different peptides a first subsetof host cells that exhibit altered ACE processing; and identifying fromthe first subset of host cells a first sub-library of the expressionlibrary comprising at least one oligonucleotide that encodes the peptidethat alters the processing of ACE.
 48. The method of claim 47, whereinthe animal host cell is a mammalian host cell.
 49. The method of claim47, wherein the animal host cell is a recombinant host cell.
 50. Themethod of claim 47, wherein the animal host cell is an isolated cell.51. The method of claim 47, wherein the first plurality of host cellsdisplay the different peptides under substantially physiologicalconditions.
 52. A method for identifying a peptide that altersprocessing of ACE membrane protein comprising: pre-enriching anexpression library comprising a plurality of oligonucleotides, at leasta majority of the oligonucleotides having different sequences encodingdifferent peptides, for at least one oligonucleotide that encodes apeptide that specifically binds to ACE, the pre-enrichment comprisingthe steps of introducing the expression library into a phage displayvector which can express the peptides encoded by the oligonucleotidesequences on the surface of the phage; expressing the different peptideson the surface of the phage; selecting a subset of phage particles thatexpress peptides that specifically bind ACE membrane protein; recoveringthe oligonucleotide sequences from the selected phage particles to forma pre-enriched expression library introducing the pre-enrichedexpression library into a first plurality of animal host cells thatexpress ACE membrane protein and at least one ACE membrane proteinprocessing enzyme, the host cells thereby expressing and displaying theat least one ACE-binding peptide within a secretory pathway and on anextracellular cell surface; selecting from the first plurality of hostcells displaying the at least one ACE membrane protein-binding peptide afirst subset of host cells that exhibit altered ACE processing; andidentifying from the first subset of host cells a first sub-library ofthe pre-enriched expression library comprising at least oneoligonucleotide that encodes the peptide that alters the processing ofACE.
 53. A method for identifying a peptide that alters processing ofACE membrane protein comprising: introducing an expression librarycomprising a plurality of oligonucleotides, at least a majority of theoligonucleotides having different sequences encoding different peptides,into a first plurality of animal host cells that express ACE membraneprotein and at least one ACE membrane protein processing enzyme, thehost cells thereby expressing and displaying the different peptideswithin a secretory pathway and on an extracellular cell surface;selecting from the first plurality of host cells displaying thedifferent peptides a first subset of host cells that exhibit altered ACEprocessing; wherein the altered ACE processing is determined byassessing the relative presence or absence of at least one species ofACE released from the surface of the host cells displaying the differentpeptides by contacting the host cells with at least one detectablylabeled marker that specifically binds to the at least one species ofACE fragment and detecting the bound labeled marker; and identifyingfrom the first subset of host cells a first sub-library of theexpression library comprising at least one oligonucleotide that encodesthe peptide that alters the processing of ACE membrane protein.
 54. Amethod for identifying a peptide that alters processing of ACE membraneprotein comprising: introducing an expression library comprising aplurality of oligonucleotides, at least a majority of theoligonucleotides having different sequences encoding different peptides,into a first plurality of animal host cells that express ACE membraneprotein and at least one ACE processing enzyme, the host cells therebyexpressing and displaying the different peptides within a secretorypathway and on an extracellular cell surface under substantiallyphysiological conditions; selecting from the first plurality of hostcells displaying the different peptides a first subset of host cellsthat exhibit altered ACE processing; wherein the altered ACE processingis determined by assessing the relative presence or absence of at leastone species of ACE fragment on the surface or released from the surfaceof the host cells displaying the different peptides by contacting thehost cells with at least one detectably labeled marker that specificallybinds to the at least one species of ACE fragment and detecting thebound labeled marker; and identifying from the first subset of hostcells a first sub-library of the expression library comprising at leastone oligonucleotide that encodes the peptide that alters the processingof ACE membrane protein.